Binding partners for the thyrotropin receptor and uses thereof

ABSTRACT

A binding partner for the TSH receptor, which binding partner comprises, or is derived from, a human monoclonal or recombinant antibody, or one or more fragments thereof, reactive with the TSH receptor, uses thereof, methods of diagnosis and therapy employing the same, and anti-idiotypic antibodies thereto.

This application is a 371 national phase of PCT/GB2003/005171, filedNov. 28, 2003, and published in English as WO 2004/050708 on Jun. 17,2004.

BACKGROUND OF THE INVENTION

The present invention is concerned with binding partners (such asmonoclonal or recombinant antibodies) for the thyrotropin receptor (TSHreceptor or TSHR) and uses thereof.

Thyrotropin or thyroid stimulating hormone (TSH) is a pituitary hormonewhich plays a key role in regulating the function of the thyroid. Itsrelease is stimulated by the hormone TRH formed in the hypothalamus andTSH controls the formation and release of the important, thyroidhormones thyroxine (T4) and tri-iodothyronine (T3). On the basis of afeedback mechanism, the thyroid hormone content of serum controls therelease of TSH. The formation of T3 and T4 by thyroid cells isstimulated by TSH by a procedure in which the TSH released by thepituitary binds to the TSH receptor of the thyroid cell membrane.

In Graves' disease (a common autoimmune disorder) TSH receptorantibodies (TRAb) are formed and these autoantibodies bind to the TSHreceptor in such a way as to mimic the actions of TSH, stimulating thethyroid gland to produce high levels of thyroid hormones. Theseautoantibodies are described as having stimulating activity. In somepatients, autoantibodies bind to the TSH receptor but do not stimulatethyroid hormone production and are described as having blockingactivity. (J Sanders, Y Oda, S-A Roberts, M Maruyama, J Furmaniak, BRees Smith; “Understanding the thyrotrophin receptor function-structurerelationship” Balliere's Clinical Endocrinology and Metabolism; Ed TFDavies 1997; 11 (3): 451-479; pub Ballière Tindall, London).

Measurements of TSH receptor antibodies are important in the diagnosisand management of Graves' disease and other thyroid disorders. Currentlythree types of assay are used to measure TSH receptor antibodies:

(a) competitive binding assays which measure the ability of TSH receptorantibodies to inhibit the binding of TSH to preparations of TSHreceptor;

(b) bioassays which measure the ability of TSH receptor antibodies tostimulate cells expressing the TSH receptor in culture; and

(c) immunoprecipitation of TSH receptor preparations with TSH receptorantibodies.

Measurement of TSH receptor antibodies using such assays are describedin references: —

J Sanders, Y Oda, S-A Roberts, M Maruyama, J Furmaniak, B Rees Smith;“Understanding the thyrotrophin receptor function-structurerelationship” Ballière's Clinical Endocrinology and Metabolism; Ed TFDavies 1997; 11 (3): 451-479; pub Ballière Tindall, London.

Sanders, Y Oda, S Roberts, A Kiddie, T Richards, J Bolton, V McGrath, SWalters, D Jaskolski, J Furmaniak, B Rees Smith; “The interaction of TSHreceptor autoantibodies with TSH receptor”; Journal of ClinicalEndocrinology and Metabolism 1999; 84 (10): 3797-3802.

It has been recognised for many years that human monoclonal antibodiesto the TSH receptor derived from patients'lymphocytes would be valuablereagents for understanding the pathogenesis of Graves' disease and fordeveloping new methods of measuring TSH receptor antibodies for exampleas replacements for TSH in competitive binding assays. Also, as thepatient's serum TSH receptor antibodies are usually powerful thyroidstimulators (TSH agonists) stimulating human monoclonal TSH receptorantibodies would be valuable for in vivo applications when tissuecontaining the TSH receptor (eg thyroid tissue or thyroid cancer tissue)required stimulation. Furthermore, as some patient serum TSH receptorantibodies are powerful TSH antagonists (blocking antibodies) humanmonoclonal TSH receptor antibodies which are TSH antagonists would bevaluable for in vivo applications when the activity of tissue containingthe TSH receptor (eg thyroid tissue or thyroid cancer tissue) requiredinactivation or to be made unresponsive to TSH, TSH receptor antibodiesor other stimulators.

It has also been recognised that one of the major advantages of humanmonoclonal TSH receptor antibodies over TSH in such in vitro and/or invivo applications would be the relative ease with which antibodies canbe manipulated. For example, manipulation of the TSH receptor bindingregion of the monoclonal antibodies so as to change theircharacteristics, such as affinity and biological characteristicsincluding their degree of TSH agonist or antagonist activities. Also,monoclonal antibodies will have a much longer half life than TSH in vivoand this may have considerable advantages in certain in vivoapplications. Furthermore, the half life of antibodies can bemanipulated easily, for example antibody Fab fragments have a muchshorter half life than intact IgG. These general properties of TSHreceptor antibodies are described in the publications such as B ReesSmith, S M McLachlan, J Furmaniak; “Autoantibodies to the thyrotropinreceptor”; Endocrine Reviews 1988; 9: 106-121; B Rees Smith, K JDorrington, D S Munro; “The thyroid stimulating properties oflong-acting thyroid stimulator yG-globulin subunits”; Biochimica etBiophysica Acta 1969; 192: 277-285; K J Dorrington, D S Munro; “The longacting thyroid stimulator”; Clinical Pharmacology and Therapeutics 1966;7: 788-806.

A still further advantage of human monoclonal TSH receptor antibodiescould be in their use to identify and provide new types of TSH receptorantibody binding sites. For example by the generation of antibodies tothe regions of the human monoclonal TSH receptor antibodies which bindthe TSH receptor. Some of the anti-idiotypic antibodies produced in thisway could have potential as new ligands for assays of TSH receptorantibodies, TSH and related compounds. Also they may be effective agentsin vivo for regulating the action of TSH receptor antibodies, TSH andrelated compounds.

Other methods of identifying and providing new types of antibody bindingsites using monoclonal antibodies are well known. For example byantibody screening of phage-displayed random peptide libraries asdescribed by J C Scott and G P Smith; “Searching for peptide ligandswith an epitope library”; Science 1990; 249 (4967): 386-390 and M AMyers, J M Davies, J C Tong, J Whisstock, M Scealy, I R MacKay, M JRowley; “Conformational epitopes on the diabetes autoantigen GAD₆₅identified by peptide phage display and molecular modelling”; Journal ofImmunology 2000; 165: 3830-3838. Antibody screening of non-peptidecompounds and libraries of non-peptide compounds can also be carriedout.

New types of TSH receptor antibody binding sites identified and providedusing these procedures may also be useful as new ligands in assays forTSH receptor antibodies, TSH and related compounds. Furthermore they maybe effective agents in vivo for regulating the action of TSH receptorantibodies, TSH and related compounds.

In view of the potential value of human monoclonal TSH receptorantibodies there have been considerable efforts over many years toproduce such antibodies (see for example B Rees Smith, S M McLachlan, JFurmaniak; “Autoantibodies to the thyrotropin receptor”; EndocrineReviews 1988; 9: 106-121. However, to date these efforts have beenunsuccessful (see for example S M McLachlan, B Rapoport; “Monoclonal,human autoantibodies to the TSH receptor—The Holy Grail and why are welooking for it”; Journal of Clinical Endocrinology and Metabolism 1996;81: 3152-3154 and J H W van der Heijden, T W A de Bruin, K A F MGludemans, J de Kruif, J P Banga, T Logtenberg; “Limitations of thesemisynthetic library approach for obtaining human monoclonalautoantibodies to the thyrotropin receptor of Graves' disease”; Clinicaland Experimental Immunology 1999; 118: 205-212).

SUMMARY OF THE INVENTION

The present provides a binding partner for TSH receptor. The bindingpartner comprises or is derived from

-   -   (a) a human monoclonal antibody reactive with the TSH receptor;    -   (b) a recombinant antibody reactive with the TSH receptor; or    -   (c) a fragment of a human monoclonal antibody or a recombinant        antibody reactive with the TSH receptor.

The binding partner of the invention can be used in therapeutic anddiagnostic applications, and for identification of epitope regions onTSH receptor.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows inhibition of TSH binding to TSH Receptor in the presenceof a binding partner of the invention. The control IgG was a humanmonoclonal antibody to GAD₆₅.

FIG. 2 shows thyroid stimulating activity of hMAb TSHR1 IgG and Fab,porcine TSH (70 units/mg; pTSH) recombinant human TSH (6.7 units/mg;hTSH) and a control monoclonal antibody (MAb; human monoclonalautoantibody to thyroid peroxidase (2G4)). Basal=cAMP produced in thepresence of NaCl free Hanks Buffered Salt Solution only.

FIG. 3A shows the effect of lymphocyte donor serum on inhibition of TSHbinding and on cAMP stimulation in TSH receptor transfected CHO cells.In the case of the binding inhibition assay the serum was diluted in apool of healthy donor sera. For the stimulation assay, the serum wasdiluted in NaCl free Hanks Buffered Salt Solution. Healthy blood donorsera (n=3) gave responses ranging from 1.1 to 1.3× basal.

FIG. 3B shows a comparison of an ELISA for TSHR autoantibodies accordingto the present invention with earlier assays: an ELISA on TSH-biotindescribed in Bolton et al., Clinical Chemistry (1999) 45: 2285-2287 andthe original RIA described by Southgate et al. in Clinical Endocrinology(1984) 20: 539-543.

FIG. 3C shows a comparison of an ELISA for TSHR autoantibodies accordingto the present invention and an ELISA based on TSH biotin as describedin Bolton et al., Clinical Chemistry (1999) 45: 2285-2287. Sera from 72patients with Graves' disease were compared. y=1.1154x−13.032, r=0.99.

FIG. 4 shows the nucleotide sequence of hMAb TSHR1 Heavy chain V, D andJ region with the primer, CDR and constant region sites labeled (Seq IDNo: 14).

FIG. 5 shows the amino acid sequence of hMAb TSHR1 Heavy chain V, D andJ region with the CDR and constant regions labeled (Seq ID No: 5).

FIG. 6 shows the nucleotide sequence of hMAb TSHR1 Light chain with theprimer and CDR sites labeled (Seq ID No: 14).

FIG. 7 shows the amino acid sequence of hMAb TSHR1 Light chain with theCDR sites labeled (Seq ID No: 6).

FIG. 8 shows effects of 2 patients sera (T1 and T2 with TSH antagonistactivity) on stimulation of cyclic AMP production by pTSH (0.5 ng/ml)and hMAb TSHR1 IgG (10 ng/ml) and Fab (5 ng/ml) in CHO cells transfectedwith the TSHR.

FIG. 9 shows the nucleotide sequence of 9D33 Heavy chain with primer,CDR and constant region sites marked. (Seq. ID No. 33).

FIG. 10 shows the amino acid sequence of 9D33 Heavy chain with primer,CDR and constant region sites marked. (Seq. ID No. 23).

FIG. 11 shows the nucleotide sequence of 9D33 light chain with primer,CDR and constant region sites marked. (Seq. ID No. 38).

FIG. 12 shows the amino acid sequence of 9D33 light chain with primer,CDR and constant region sites marked. (Seq. ID No. 28).

DETAILED DESCRIPTION OF THE INVENTION

It is an object of the present invention to provide a binding partnerfor the TSH receptor capable of interacting with the TSH receptor in amanner comparable to the interaction of TSH receptor autoantibodies withthe TSH receptor, in particular it is an object of the present inventionto provide human monoclonal antibodies to the TSH receptor exhibiting acomparable interaction therewith as seen with TSH receptor antibodiespresent in the sera of patients with hyperthyroid Graves' disease andalso to provide recombinant preparations thereof. The considerabledifficulties of producing human monoclonal TSH receptor antibodies havebeen overcome in the invention described herein. In particular thesuccessful production of a human monoclonal TSH receptor antibody withthe characteristics of the autoantibodies found in the sera of patientswith hyperthyroid Graves' disease is described. The human TSH receptormonoclonal antibody we have produced (described herein as hMAb TSHR 1)binds to the TSH receptor with high affinity and in such a way thatsmall amounts of the antibody inhibit labelled TSH binding to the TSHreceptor and small amounts act as powerful thyroid stimulators. Fabfragments of the antibody and recombinant Fab preparations are similarlyeffective thyroid stimulators and inhibitors of labelled TSH binding asintact IgG. Monoclonal Fab and/or intact IgG can be labelled with ¹²⁵Ior biotin and shown to bind to the TSH receptor. Such binding isinhibited by TSH receptor autoantibodies in patient sera.

There is provided by the present invention, therefore, a binding partnerfor the TSH receptor, which binding partner comprises, or is derivedfrom, a human monoclonal or recombinant antibody, or one or morefragments thereof, reactive with the TSH receptor.

In particular, there is provided by the present invention a bindingpartner for the TSH receptor, which binding partner comprises, or isderived from, a human monoclonal antibody, or one or more fragmentsthereof, reactive with the TSH receptor.

In particular, there is provided by the present invention a bindingpartner for the TSH receptor, which binding partner comprises, or isderived from, a human recombinant antibody, or one or more fragmentsthereof, reactive with the TSH receptor.

In particular, there is provided by the present invention a humanmonoclonal antibody, or one or more fragments thereof, reactive with theTSH receptor.

In particular, there is provided by the present invention a humanrecombinant antibody, or one or more fragments thereof, reactive withthe TSH receptor. Particularly, the present invention provides one ormore fragments of a human recombinant antibody reactive with the TSHreceptor.

A binding partner according to the present invention, and in particular,a human monoclonal or recombinant antibody reactive with the TSHreceptor according to the present invention can be further characterisedby its ability to inhibit TSH binding to the TSH receptor, and/or itsability to stimulate the TSH receptor, both of which have been seen tobe comparable to the respective inhibitory and stimulatory properties ofTSH receptor autoantibodies present in sera obtained from patients withGraves' disease.

More particularly, a binding partner according to the present invention,and in particular a human monoclonal or recombinant antibody accordingto the present invention, can be characterised by an inhibitory activitywith respect to TSH binding to the TSH receptor, of at least about 15units of International Standard NIBSC 90/672 per mg, more preferably ofat least about 30 units of International Standard NIBSC 90/672 per mg,more preferably of at least about 60 units of International StandardNIBSC 90/672 per mg, or more preferably of at least about 120 units ofInternational Standard NIBSC 90/672 per mg, or one or more fragments ofsuch a monoclonal or recombinant antibody.

More particularly, a binding partner according to the present invention,and in particular a human monoclonal or recombinant antibody accordingto the present invention, can be further characterised by a stimulatoryactivity with respect to cAMP production by cells expressing the TSHreceptor, of at least about 30 units of International Standard NIBSC90/672 per mg, more preferably of at least about 60 units ofInternational Standard NIBSC 90/672 per mg, more preferably of at leastabout 120 units of International Standard NIBSC 90/672 per mg, or morepreferably of at least about 240 units of International Standard NIBSC90/672 per mg, or one or more fragments of such a monoclonal orrecombinant antibody.

In a preferred embodiment of the present invention, a binding partneraccording to the present invention, and in particular a human monoclonalor recombinant antibody according to the present invention, can becharacterised by:

-   -   (i) an inhibitory activity with respect to TSH binding to the        TSH receptor, of at least about 15 units of International        Standard NIBSC 90/672 per mg, more preferably of at least about        30 units of International Standard NIBSC 90/672 per mg, more        preferably of at least about 60 units of International Standard        NIBSC 90/672 per mg, or more preferably of at least about 120        units of International Standard NIBSC 90/672 per mg; and    -   (ii) a stimulatory activity with respect to cAMP production by        cells expressing the TSH receptor, of at least about 30 units of        International Standard NIBSC 90/672 per mg, more preferably of        at leas about 60 units of International Standard NIBSC 90/672        per mg, more preferably of at least about 120 units of        International Standard NIBSC 90/672 per mg, or more preferably        of at least about 240 units of International Standard NIBSC        90/672 per mg;        or one or more fragments of such a monoclonal or recombinant        antibody.

In the case where a binding partner according to the present inventioncomprises or is derived from one or more fragments of a monoclonal orrecombinant antibody reactive with the TSH receptor, in particular forexample one or more Fab fragments of a monoclonal or recombinantantibody reactive with the TSH receptor, it may be preferred that such abinding partner can be characterised by an inhibitory activity withrespect to TSH binding to the TSH receptor, of at least about 30 unitsof International Standard NIBSC 90/672 per mg, more preferably of atleast about 60 units of International Standard NIBSC 90/672 per mg, morepreferably of at least about 120 units of International Standard NIBSC90/672 per mg, or more preferably of at least about 240 units ofInternational Standard NIBSC 90/672 per mg.

It may also be preferred in the case where a binding partner accordingto the present invention comprises or is derived from one or morefragments of a monoclonal or recombinant antibody reactive with the TSHreceptor, in particular for example one or more Fab fragments of amonoclonal or recombinant antibody reactive with the TSH receptor, thatsuch a binding partner can be characterised by a stimulatory activitywith respect to cAMP production by cells expressing the TSH receptor, ofat least about 50 units of International Standard NIBSC 90/672 per mg,more preferably of at least about 100 units of International StandardNIBSC 90/672 per mg, more preferably of at least about 200 units ofInternational Standard NIBSC 90/672 per mg, or more preferably of atleast about 400 units of International Standard NIBSC 90/672 per mg.

It may be still further preferred in the case where a binding partneraccording to the present invention comprises or is derived from one ormore fragments of a monoclonal or recombinant antibody reactive with theTSH receptor, in particular for example one or more Fab fragments of amonoclonal or recombinant antibody reactive with the TSH receptor, thatsuch a binding partner can be characterised by:

-   -   (i) an inhibitory activity with respect to TSH binding to the        TSH receptor, of at least about 30 units of International        Standard NIBSC 90/672 per mg, more preferably of at least about        60 units of International Standard NIBSC 90/672 per mg, more        preferably of at least about 120 units of International Standard        NIBSC 90/672 per mg, or more preferably of at least about 240        units of International Standard NIBSC 90/672 per mg; and    -   (ii) a stimulatory activity with respect to cAMP production by        cells expressing the TSH receptor, of at least about 50 units of        International Standard NIBSC 90/672 per mg, more preferably of        at least about 100 units of International Standard NIBSC 90/672        per mg, more preferably of at least about 200 units of        International Standard NIBSC 90/672 per mg, or more preferably        of at least about 400 units of International Standard NIBSC        90/672 per mg.

In a preferred case the present invention provides a binding partner forthe TSH receptor (typically a human monoclonal antibody), which bindingpartner is capable of binding to the TSH receptor preferably so as tostimulate the TSH receptor and which comprises an antibody V_(H) domainselected from the group consisting of a V_(H) domain as shown in SEQ IDNO. 1 and a V_(H) domain comprising one or more V_(H) CDRs with an aminoacid sequence selected from SEQ ID NO. 2, SEQ ID NO. 3 and SEQ ID NO. 4.

In a first embodiment of the present invention, there is, therefore,provided a binding partner for the TSH receptor (typically a humanmonoclonal antibody), which binding partner is capable of binding to theTSH receptor preferably so as to stimulate the TSH receptor and whichcomprises an antibody V_(H) domain as shown in SEQ ID NO. 1.

In a second embodiment of the present invention there is, therefore,provided a binding partner for the TSH receptor (typically a humanmonoclonal antibody), which binding partner is capable of binding to theTSH receptor preferably so as to stimulate the TSH receptor and whichcomprises an antibody V_(H) domain comprising one or more V_(H) CDRswith an amino acid sequence selected from SEQ ID NO. 2, SEQ ID NO. 3 andSEQ ID NO. 4.

It will be appreciated that a binding partner according to the presentinvention can comprise an antibody V_(H) domain substantially ashereinbefore described in the absence of an antibody V_(L) domain. It isknown that single immunoglobulin domains, especially V_(H) domains, arecapable of binding target antigens in a specific manner. Alternatively,a binding partner according to the present invention can comprise anantibody V_(H) domain paired with an antibody V_(L) domain to provide anantibody binding site comprising both V_(H) and V_(L) domains for a TSHreceptor employing techniques well known in the art (Biochim. Biophys.Acta, 192 (1969) 277-285; Proc. Natl. Acad. Sci. USA, Vol. 89, pp10026-10030, November 1992).

In a preferred case the present invention provides, however, a bindingpartner for the TSH receptor, which binding partner is capable ofbinding to the TSH receptor preferably so as to stimulate the TSHreceptor and which comprises:

-   an antibody V_(H) domain selected from the group consisting of:    -   a V_(H) domain as shown in SEQ ID NO. 1 and a V_(H) domain        comprising one or more V_(H) CDRs with an amino acid sequence        selected from SEQ ID NO. 2, SEQ ID NO. 3 and SEQ ID NO. 4;        and/or-   an antibody V_(L) domain selected from the group consisting of:    -   a V_(L) domain as shown in SEQ ID NO. 6 and a V_(L) domain        comprising one or more V_(L) CDRs with an amino acid sequence        selected from SEQ ID NO. 7, SEQ ID NO. 8 and SEQ ID NO. 9.

It may be preferred according to the present invention that a bindingpartner substantially as hereinbefore described comprises an antibodyV_(H) domain substantially as hereinbefore described paired with anantibody V_(L) domain substantially as hereinbefore described to providean antibody binding site comprising both V_(H) and V_(L) domains for theTSH receptor, although as discussed further an antibody V_(H) domain, oran antibody V_(L) domain, may be independently used to bind a TSHreceptor. It will be appreciated, therefore, that a binding partnersubstantially as hereinbefore described can comprise an antibody V_(H)domain substantially as hereinbefore described in the absence of anantibody V_(L) domain. It will also be appreciated, therefore, that abinding partner substantially as hereinbefore described can comprise anantibody V_(L) domain substantially as hereinbefore described in theabsence of an antibody V_(H) domain. Alternatively, a binding partnersubstantially as hereinbefore described can comprise an antibody V_(H)domain paired with an antibody V_(L) domain substantially ashereinbefore described to provide an antibody binding site comprisingboth V_(H) and V_(L) domains for the TSH receptor.

Preferred embodiments according to the present invention can thusinclude a binding partner substantially as hereinbefore describedcomprising an antibody V_(H) domain as shown in SEQ ID NO. 1 paired withan antibody V_(L) domain as shown in SEQ ID NO. 6 to provide an antibodybinding site, comprising both these V_(H) and V_(L) domains for the TSHreceptor.

It is further envisaged according to the present invention that V_(H)domains substantially as hereinbefore described may be paired with V_(L)domains other than those specifically described herein. It is alsofurther envisaged according to the present invention that V_(L) domainssubstantially as hereinbefore described may be paired with V_(H) domainsother than those specifically described herein.

According to a further embodiment of the present invention there isprovided a binding partner substantially as hereinbefore described forthe TSH receptor, which binding partner is capable of binding to the TSHreceptor so as to stimulate the TSH receptor and which can comprise:

-   an antibody V_(H) domain comprising:    -   a V_(H) domain comprising one or more V_(H) CDRs with an amino        acid sequence selected from SEQ ID NO. 2, SEQ ID NO. 3 and SEQ        ID NO. 4; and/or-   an antibody V_(L) domain comprising:    -   a V_(L) domain comprising one or more V_(L) CDRs with an amino        acid sequence selected from SEQ ID NO. 7, SEQ ID NO. 8 and SEQ        ID NO. 9.

One or more CDRs as referred to above may be taken from the hereinbeforedescribed V_(H) and V_(L) domains and incorporated into a suitableframework. For example, the amino acid sequence of one or more CDRssubstantially as hereinbefore described may be incorporated intoframework regions of antibodies differing from hMAb TSHR 1 specificallydisclosed herein, such antibodies thereby incorporating the one or moreCDRs and being capable of binding to the TSH receptor, preferably tostimulate the TSH receptor substantially as hereinbefore described.Alternatively, the present invention may provide a polypeptide capableof binding to the TSH receptor so as to stimulate the TSH receptorsubstantially as hereinbefore described and comprising the primarystructural conformation of amino acids as represented by one or moreCDRs as specifically described herein, optionally together with furtheramino acids, which further amino acids may enhance the binding affinityof one or more CDRs as described herein for the TSH receptor or may havesubstantially no role in affecting the binding properties of thepolypeptide for the TSH receptor.

The present invention, also encompasses variants, analogs, derivativesand fragments of the specific human monoclonal antibody describedherein, V_(H) domains, CDRs and polypeptides disclosed herein, whichvariants, analogs, derivatives and fragments retain the ability tointeract with the TSH receptor (such as for example to stimulate the TSHreceptor) substantially as hereinbefore described.

The terms “variants”, “analogs”, “derivatives” and “fragments” as usedherein can be characterised as antibodies, antibody fragments orpolypeptides which retain essentially the same biological function oractivity as a human monoclonal antibody having a V_(H) domain as shownin SEQ ID NO. 1 and a V_(L) domain as shown in SEQ ID NO. 6 and inparticular in respect of the binding properties thereof for the TSHreceptor. Suitably, variants, analogs, derivatives and fragments, andvariants, analogs and derivatives of the fragments as described herein,have a primary structural conformation of amino acids in which severalor a few (such as 5 to 10, 1 to 5 or 1 to 3) amino acid residues of ahuman monoclonal antibody having a V_(H) domain as shown in SEQ ID NO. 1and a V_(L) domain as shown in SEQ ID NO. 6 are substituted, deleted oradded, in any combination. Especially preferred among these are silentsubstitutions, additions and deletions which do not alter orsubstantially alter the biological activity or function of a humanmonoclonal antibody having a V_(H) domain as shown in SEQ ID NO. 1 and aV_(L) domain as shown in SEQ ID NO. 6. Conservative substitutions can bepreferred as hereinafter described in greater detail.

More particularly, variants, analogs or derivatives of a humanmonoclonal antibody having a V_(H) domain as shown in SEQ ID NO. 1 and aV_(L) domain as shown in SEQ ID NO. 6 according to the present inventionmay be ones in which one or more of the amino acid residues aresubstituted with a conserved or non-conserved amino acid residue(preferably a conserved amino acid residue), or ones in which one ormore of the amino acid resides includes a substituent group or the like.Such variants, derivatives and analogs are deemed to be within the scopeof those skilled in the art from the teachings herein.

Most typically, variants, analogs or derivatives are those that varyfrom a reference human monoclonal antibody having a V_(H) domain asshown in SEQ ID NO. 1 and a V_(L) domain as shown in SEQ ID NO. 6 byconservative amino acid substitutions. Such substitutions are those thatsubstitute a given amino acid by another amino acid of likecharacteristics. Typically seen as conservative substitutions are thereplacements, one for another, among the aliphatic amino acids A, V, Land I; among the hydroxyl residues S and T; among the acidic residues Dand E; among the amide residues N and Q; among the basic residues K andR; and among the aromatic residues F and Y.

It will be appreciated that the term fragment as used herein inparticular relates to fragments of antibodies specifically as hereindescribed and form an important aspect of the present invention.

In this way, a human monoclonal or recombinant antibody as provided bythe present invention may be provided as any of the following fragments:(i) the Fab fragment consisting of V_(L), V_(H), C_(L) and C_(H) 1domains; (ii) the Fd fragment consisting of the V_(H) and C_(H) 1domains; (iii) the Fv fragment consisting of the V_(L) and V_(H)domains; (iv) the dAb fragment which consists of a V_(H) domain; (v)isolated CDR regions; (vi) F (ab′) 2 fragments, a bivalent fragmentcomprising two linked Fab fragments; and (vii) single chain Fv molecules(scFv), wherein a V_(H) domain and a V_(L) domain are linked by apeptide linker which allows the two domains to associate to form anantigen binding site.

Alternatively, a human monoclonal or recombinant antibody according tothe present invention may comprise a whole IgG antibody, whereby theantibody includes variable and constant regions.

The present invention also provides a further binding partner capable ofbinding to the TSH receptor, which can compete for binding to the TSHreceptor with a binding partner for the TSH receptor (typically a humanmonoclonal antibody) substantially as hereinbefore described, whichfurther binding partner does not comprise TSH. Preferably, this furtherbinding partner may comprise a further antibody having a binding sitefor an epitope region of the TSH receptor, and which can compete forbinding to the TSH receptor with a binding partner for the TSH receptor(typically a human monoclonal antibody) substantially as hereinbeforedescribed. A suitable such further binding partner can comprise a mousemonoclonal antibody, which can preferably be produced according totechniques substantially as described in the Examples, employingimmunisation of mice with TSH receptor by techniques known in the art.

The present invention may also provide a further binding partner capableof binding to the TSH receptor, which can comprise, or is derived from,a human monoclonal or recombinant antibody, or one or more fragmentsthereof, reactive with the TSH receptor. In particular this furtherbinding partner may comprise a further antibody having a binding sitefor an epitope region of the TSH receptor, which further antibody iscapable of binding to the TSH receptor, and can compete for binding tothe TSH receptor with a binding partner for the TSH receptor (typicallya human monoclonal antibody) substantially as hereinbefore described.Suitably such a further binding partner can be derived from a specificbinding partner as described herein, hMAb TSHR 1, by suitablemutagenesis techniques, such as spot mutations or the like, so as toobtain a further binding partner for the TSH receptor that can competewith a binding partner substantially as herein described (such as hMAbTSHR 1) for interaction with the TSH receptor.

Preferably a further binding partner for the TSH receptor can comprise amonoclonal or recombinant antibody and can be characterised by aninhibitory activity with respect to TSH binding to the TSH receptor, ofat least about 15 units of International Standard NIBSC 90/672 per mg,more preferably of at least about 30 units of International StandardNIBSC 90/672 per mg, more preferably of at least about 60 units ofInternational Standard NIBSC 90/672 per mg, or more preferably of atleast about 120 units of International Standard NIBSC 90/672 per mg, orone or more fragments of the antibody. It may also be preferred thatsuch a further binding partner according to the present invention, canbe characterised by a stimulatory activity with respect to cAMPproduction by cells expressing the TSH receptor, of at least about 30units of International Standard NIBSC 90/672 per mg, more preferably ofat least about 60 units of International Standard NIBSC 90/672 per mg,more preferably of at least about 120 units of International StandardNIBSC 90/672 per mg, or more preferably of at least about 240 units ofInternational Standard NIBSC 90/672 per mg, or one or more fragments ofthe antibody.

It may also be even more preferred that such a further binding partnerof the present invention, can be characterised by:

-   -   (i) an inhibitory activity with respect to TSH binding to the        TSH receptor, of at least about 15 units of International        Standard NIBSC 90/672 per mg, more preferably of at least about        30 units of International Standard NIBSC 90/672 per mg, more        preferably of at least about 60 units of International Standard        NIBSC 90/672 per mg, or more preferably of at least about 120        units of International Standard NIBSC 90/672 per mg; and    -   (ii) a stimulatory activity with respect to cAMP production by        cells expressing the TSH receptor, of at least about 30 units of        International Standard NIBSC 90/672 per mg, more preferably of        at least about 60 units of International Standard NIBSC 90/672        per mg, more preferably of at least about 120 units of        International Standard NIBSC 90/672 per mg, or more preferably        of at least about 240 units of International Standard NIBSC        90/672 per mg;        or one or more fragments thereof.

A preferred mouse monoclonal antibody providing a further bindingpartner according to the present invention comprises 9D33 preparedfurther to the Examples and having amino acid and polynucleotidesequences as illustrated by FIGS. 9 to 12 and Sequence Listings 19 to38. According to the present invention, there is, therefore, provided afurther binding partner for the TSH receptor (typically a mousemonoclonal antibody), which comprises an antibody V_(H) domain as shownin SEQ ID NO. 19.

A further binding partner as provided by the present invention can alsobe characterised as comprising an antibody V_(H) domain comprising oneor more V_(H) CDRs with an amino acid sequence selected from SEQ ID NO.20, SEQ ID NO. 21 and SEQ ID NO. 22.

It will be appreciated that a further binding partner according to thepresent invention can comprise an antibody V_(H) domain substantially ashereinbefore described in the absence of an antibody V_(L) domain. It isknown that single immunoglobulin domains, especially V_(H) domains, arecapable of binding target antigens in a specific manner. Alternatively,a further binding partner according to the present invention cancomprise an antibody V_(H) domain paired with an antibody V_(L) domainto provide an antibody binding site comprising both V_(H) and V_(L)domains for a TSH receptor employing techniques well known in the art(Biochim. Biophys. Acta, 192 (1969) 277-285; Proc. Natl. Acad. Sci. USA,Vol. 89, pp 10026-10030, November 1992).

In a preferred case the present invention provides, however, a furtherbinding partner for the TSH receptor, which further binding partnercomprises:

-   an antibody V_(H) domain selected from the group consisting of:    -   a V_(H) domain as shown in SEQ ID NO. 19 and a V_(H) domain        comprising one or more V_(H) CDRs with an amino acid sequence        selected from SEQ ID NO. 20, SEQ ID NO. 21 and SEQ ID NO. 22;        and/or-   an antibody V_(L) domain selected from the group consisting of:    -   a V_(L) domain as shown in SEQ ID NO. 24 and a V_(L) domain        comprising one or more V_(L) CDRs with an amino acid sequence        selected from SEQ ID NO. 25, SEQ ID NO. 26 and SEQ ID NO. 27.

It may be preferred according to the present invention that a furtherbinding partner substantially as hereinbefore described comprises anantibody V_(H) domain substantially as hereinbefore described pairedwith an antibody V_(L) domain substantially as hereinbefore described toprovide an antibody binding site comprising both V_(H) and V_(L) domainsfor the TSH receptor, although as discussed further an antibody V_(H)domain, or an antibody V_(L) domain, may be independently used to bind aTSH receptor. It will be appreciated, therefore, that a further bindingpartner substantially as hereinbefore described can comprise an antibodyV_(H) domain substantially as hereinbefore described in the absence ofan antibody V_(L) domain. It will also be appreciated, therefore, that afurther binding partner substantially as hereinbefore described cancomprise an antibody V_(L) domain substantially as hereinbeforedescribed in the absence of an antibody V_(H) domain. Alternatively, afurther binding partner substantially as hereinbefore described cancomprise an antibody V_(H) domain paired with an antibody V_(L) domainsubstantially as hereinbefore described to provide an antibody bindingsite comprising both V_(H) and V_(L) domains for the TSH receptor.

Preferred embodiments according to the present invention can thusinclude a further binding partner substantially as hereinbeforedescribed comprising an antibody V_(H) domain as shown in SEQ ID NO. 19paired with an antibody V_(L) domain as shown in SEQ ID NO. 24 toprovide an antibody binding site, comprising both these V_(H) and V_(L)domains for the TSH receptor.

It is further envisaged according to the present invention that V_(H)domains substantially as hereinbefore described may be paired with V_(L)domains other than those specifically described herein. It is alsofurther envisaged according to the present invention that V_(L) domainssubstantially as hereinbefore described may be paired with V_(H) domainsother than those specifically described herein.

According to a further embodiment of the present invention there isprovided a further binding partner substantially as hereinbeforedescribed for the TSH receptor, which further binding partner is capableof binding to the TSH receptor so as to inhibit stimulation of the TSHreceptor and which can comprise:

-   an antibody V_(H) domain comprising:    -   a V_(H) domain comprising one or more V_(H) CDRs with an amino        acid sequence selected from SEQ ID NO. 20, SEQ ID NO. 21 and SEQ        ID NO. 22; and/or-   an antibody V_(L) domain comprising:    -   a V_(L) domain comprising one or more V_(L) CDRs with an amino        acid sequence selected from SEQ ID NO. 25, SEQ ID NO. 26 and SEQ        ID NO. 27.

One or more CDRs as referred to above may be taken from the hereinbeforedescribed V_(H) and V_(L) domains and incorporated into a suitableframework. For example, the amino acid sequence of one or more CDRssubstantially as hereinbefore described may be incorporated intoframework regions of antibodies differing from 9D33 specificallydisclosed herein, such antibodies thereby incorporating the one or moreCDRs and being capable of binding to the TSH receptor. Alternatively,the present invention may provide a polypeptide capable of binding tothe TSH receptor comprising the primary structural conformation of aminoacids as represented by one or more CDRs as specifically describedherein, optionally together with further amino acids, which furtheramino acids may enhance the binding affinity of one or more CDRs asdescribed herein for the TSH receptor or may have substantially no rolein affecting the binding properties of the polypeptide for the TSHreceptor.

It will be appreciated that the term fragment as used herein inparticular relates to fragments of antibodies specifically as hereindescribed and form an important aspect of the present invention. In thisway, a further binding partner according to the present invention may beprovided as any of the following fragments: (i) the Fab fragmentconsisting of V_(L), V_(H), C_(L) and C_(H) 1 domains; (ii) the Fdfragment consisting of the V_(H) and C_(H) 1 domains; (iii) the Fvfragment consisting of the V_(L) and V_(H) domains; (iv) the dAbfragment which consists of a V_(H) domain; (v) isolated CDR regions;(vi) F (ab′) 2 fragments, a bivalent fragment comprising two linked Fabfragments; and (vii) single chain Fv molecules (scFv), wherein a V_(H)domain and a V_(L) domain are linked by a peptide linker which allowsthe two domains to associate to form an antigen binding site.

Alternatively, a mouse monoclonal or recombinant antibody according tothe present invention, such as 9D33, may comprise a whole IgG antibody,whereby the antibody includes variable and constant regions.

There is also provided by the present invention a polynucleotidecomprising:

-   -   (i) a nucleotide sequence as shown in SEQ ID NO. 10, SEQ ID NO.        11, SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO. 16,        SEQ ID NO. 17 or SEQ ID NO. 18, encoding an amino acid sequence        of an antibody V_(H) domain, V_(L) domain, or CDR, as shown in        SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID        NO. 6, SEQ ID NO. 7, SEQ ID NO. 8 or SEQ ID NO. 9;    -   (ii) a nucleotide sequence encoding a binding partner for the        TSH receptor (typically a human monoclonal antibody)        substantially as hereinbefore described, or encoding an amino        acid sequence of an antibody V_(H) domain, V_(L) domain, or CDR,        of a binding partner for the TSH receptor (typically a human        monoclonal antibody) substantially as hereinbefore described;    -   (iii) a nucleotide sequence differing from any sequence of (i)        in codon sequence due to the degeneracy of the genetic code;    -   (iv) a nucleotide sequence comprising an allelic variation of        any sequence of (i);    -   (v) a nucleotide sequence comprising a fragment of any of the        sequences of (i), (ii), (iii), or (iv) and in particular a        nucleotide sequence comprising a fragment of any of the        sequences of (i), (ii), (iii), (iv) or (v) and encoding a Fab        fragment, a Fd fragment, a Fv fragment, a dAb fragment, an        isolated CDR region, F (ab′) 2 fragments or a scFv fragment, of        a human monoclonal antibody substantially as hereinbefore        described;    -   (vi) a nucleotide sequence differing from the any sequence        of (i) due to mutation, deletion or substitution of a nucleotide        base and encoding a binding partner for the TSH receptor        (typically a human monoclonal antibody) substantially as        hereinbefore described, or encoding an amino acid sequence of an        antibody V_(H) domain, V_(L) domain, or CDR, of a binding        partner for the TSH receptor (typically a human monoclonal        antibody) substantially as hereinbefore described.

There is also provided by the present invention a polynucleotidecomprising:

-   -   (i) a nucleotide sequence as shown in SEQ ID NO. 29, SEQ ID NO.        30, SEQ ID NO. 31, SEQ ID NO. 32, SEQ ID NO. 34, SEQ ID NO. 35,        SEQ ID NO. 36 or SEQ ID NO. 37, encoding an amino acid sequence        of an antibody V_(H) domain, V_(L) domain, or CDR, as shown in        SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22, SEQ        ID NO. 24, SEQ ID NO. 25, SEQ ID NO. 26 or SEQ ID NO. 27;    -   (ii) a nucleotide sequence encoding a further binding partner        for the TSH receptor (typically a mouse monoclonal antibody)        substantially as hereinbefore described, or encoding an amino        acid sequence of an antibody V_(H) domain, V_(L) domain, or CDR,        of a further binding partner for the TSH receptor (typically a        mouse monoclonal antibody) substantially as hereinbefore        described;    -   (iii) a nucleotide sequence differing from any sequence of (i)        in codon sequence due to the degeneracy of the genetic code;    -   (iv) a nucleotide sequence comprising an allelic variation of        any sequence of (i);    -   (v) a nucleotide sequence comprising a fragment of any of the        sequences of (i), (ii), (iii), or (iv) and in particular a        nucleotide sequence comprising a fragment of any of the        sequences of (i), (ii), (iii), (iv) or (v) and encoding a Fab        fragment, a Fd fragment, a Fv fragment, a dAb fragment, an        isolated CDR region, F (ab′) 2 fragments or a scFv fragment, of        a mouse monoclonal antibody substantially as hereinbefore        described;    -   (vi) a nucleotide sequence differing from the any sequence        of (i) due to mutation, deletion or substitution of a nucleotide        base and encoding a further binding partner for the TSH receptor        (typically a mouse monoclonal antibody) substantially as        hereinbefore described, or encoding an amino acid sequence of an        antibody V_(H) domain, V_(L) domain, or CDR, of a further        binding partner for the TSH receptor (typically a mouse        monoclonal antibody) substantially as hereinbefore described.

Variant polynucleotides according to the present invention are suitablyat least 70% identical over their entire length to any polynucleotidesequence of (i), most highly preferred are polynucleotides that comprisea region that is at least 80% identical over its entire length to anypolynucleotide sequence of (i), polynucleotides at least 90% identicalover their entire length to any polynucleotide sequence of (i) areparticularly preferred, and among these particularly preferredpolynucleotides, those with at least 95% identity are especiallypreferred.

The present invention further provides a biologically functional vectorsystem which carries a polynucleotide substantially as hereinbeforedescribed and which is capable of introducing the polynucleotide intothe genome of a host organism.

The present invention also relates to host cells which are transformedwith polynucleotides of the invention and the production of bindingpartners for the TSH receptor of the invention by recombinanttechniques. Host cells can be genetically engineered to incorporatepolynucleotides and express binding partners for the TSH receptor of thepresent invention.

The amino acid sequences of hMAb TSHR 1, a human monoclonal antibodyaccording to the present invention, and nucleotide sequences codingtherefor, the amino acid sequences of 9D33, a mouse monoclonal antibodywhich represents a further binding partner according to the presentinvention, and nucleotide sequences coding therefor, are shown in theSequence listings as herein after described and can be assigned asfollows.

For hMAb TSHR 1:

Amino Acid Sequences

SEQ ID NO. 1 V_(H)

SEQ ID NO. 2 V_(H) CDRI

SEQ ID NO. 3 V_(H) CDRII

SEQ ID NO. 4 V_(H) CDRIII

SEQ ID NO. 5 Heavy chain variable and adjacent constant region

SEQ ID NO. 6 V_(L)

SEQ ID NO. 7 V_(L) CDRI

SEQ ID NO. 8 V_(L) CDRII

SEQ ID NO. 9 V_(L) CDRIII

Nucleotide Sequences

SEQ ID NO. 10 V_(H)

SEQ ID NO. 11 V_(H) CDRI

SEQ ID NO. 12 V_(H) CDRII

SEQ ID NO. 13 V_(H) CDRIII.

SEQ ID NO. 14 Heavy chain variable and adjacent constant region

SEQ ID NO. 15 V_(L)

SEQ ID NO. 16 V_(L) CDRI

SEQ ID NO. 17 V_(L) CDRII

SEQ ID NO. 18 V_(L) CDRIII

For 9D33:

Amino Acid Sequences

SEQ ID NO. 19 V_(H)

SEQ ID NO. 20 V_(H) CDRI

SEQ ID NO. 21 V_(H) CDRII

SEQ ID NO. 22 V_(H) CDRIII

SEQ ID NO. 23 Heavy chain variable and adjacent constant region

SEQ ID NO. 24 V_(L)

SEQ ID NO. 25 V_(L) CDRI

SEQ ID NO. 26 V_(L) CDRII

SEQ ID NO. 27 V_(L) CDRIII

SEQ ID NO. 28 Light chain variable and adjacent constant region

Nucleotide Sequences

SEQ ID NO. 29 V_(H)

SEQ ID NO. 30 V_(H) CDRI

SEQ ID NO. 31 V_(H) CDRII

SEQ ID NO. 32 V_(H) CDRIII

SEQ ID NO. 33 Heavy chain variable and adjacent constant region

SEQ ID NO. 34 V_(L)

SEQ ID NO. 35 V_(L) CDRI

SEQ ID NO. 36 V_(L) CDRII

SEQ ID NO. 37 V_(L) CDRIII

SEQ ID NO. 38 Light chain variable and adjacent constant region

The above sequences for hMab TSHR1 can also be seen by reference toFIGS. 4, 5, 6 and 7, wherein:

FIG. 4 shows the hMab TSHR1 heavy chain nucleotide sequence, along withthe adjacent constant region, annotated with the PCR primer, CDRI,CDRII, CDRIII and constant regions;

FIG. 5 shows the hMab TSHR1 heavy chain amino acid sequence, along withthe adjacent constant region, annotated with the CDRI, CDRII, CDRIII andconstant regions;

FIG. 6 shows the hMab TSHR1 light chain nucleotide sequence annotatedwith the PCR primer, CDRI, CDRII and CDRIII regions; and

FIG. 7 shows the hMab TSHR1 light chain amino acid sequence annotatedwith the CDRI, CDRII and CDRIII regions.

It will be appreciated from the above that for the V_(H) chain of hMabTSHR1 the nucleotide sequences of the CDRI, CDRII and CDRIII regions asshown in FIG. 4 correspond to the V_(H)CDRI., V_(H) CDRII and V_(H)CDRIII sequences shown in SEQ ID NO.s 11, 12 and 13 respectively, andthat the amino acid sequences of the CDRI, CDRII and CDRIII regions asshown in FIG. 5 correspond to the V_(H) CDRI, V_(H) CDRII and V_(H)CDRIII sequences shown in SEQ ID NO.s 2,3 and 4 respectively. It willalso be appreciated from the above that for the V_(L) chain of hMabTSHR1 the nucleotide sequences of the CDRI, CDRII and CDRIII regions asshown in FIG. 6 correspond to the V_(L) CDRI, V_(L) CDRII and V_(L)CDRIII sequences shown in SEQ ID NO.s 16,17 and 18 respectively, andthat the amino acid sequences of the CDRI, CDRII and CDRIII regions asshown in FIG. 7 correspond to the V_(L) CDRI, V_(L) CDRII and V_(L)CDRIII sequences shown in SEQ ID NO.s 7,8 and 9 respectively.

Analysis of the crystal structure of hMAb TSHRI Fab (determined bytechniques known in the art) enabled refinement of the HC and LCnucleotide sequences determined using PCR primers which are degenerate.In particular, a HC sequencing artefact for nucleotides 115-120 wasidentified. Sequencing indicated cacgtg (transcribed to amino acids HisVal), whereas the crystal structure more reliably indicated amino acidsGln Leu (corresponding bases being cagctg), with the refined sequencesbeing shown in the accompanying Figures and Sequence listings. Crystalstructure analysis also enabled refinement of the HC and LC derivedamino acid sequences particularly in the degenerate PCR primer region.In the case of the LC aa 2 was found to be Pro by RT-PCR but was Thrfrom the crystal structure. In the case of the HC aa 2 was found to beMet by RT-PCR but was Val from the crystal structure. Again, theserefined sequences are shown in the accompanying Figures and Sequencelistings.

The above sequences for 9D33 can also be seen by reference to FIGS. 9,10, 11 and 12, wherein:

FIG. 9 shows the 9D33 heavy chain nucleotide sequence, along with theadjacent constant region, annotated with the PCR primer, CDRI, CDRII,CDRIII and constant regions;

FIG. 10 shows the 9D33 heavy chain amino acid sequence, along with theadjacent constant region, annotated with the PCR primer, CDRI, CDRII,CDRIII and constant regions;

FIG. 11 shows the 9D33 light chain nucleotide sequence annotated withthe PCR primer, CDRI, CDRII, CDRIII and constant regions;

FIG. 12 shows the 9D33 light chain amino acid sequence annotated withthe PCR primer, CDRI, CDRII, CDRIII and constant regions.

It will be appreciated from the above that for the V_(H) chain of 9D33the nucleotide sequences of the CDRI, CDRII and CDRIII regions as shownin FIG. 9 correspond to the V_(H) CDRI, V_(H)CDRII and V_(H) CDRIIIsequences shown in SEQ ID NO.s 30,31 and 32 respectively, and that theamino acid sequences of the CDRI, CDRII and CDRIII regions as shown inFIG. 10 correspond to the V_(H) CDRI, V_(H) CDRII and V_(H) CDRIIIsequences shown in SEQ ID NO.s 20, 21 and 22 respectively. It will alsobe appreciated from the above that for the V_(L) chain of 9D33 thenucleotide sequences of the CDRI, CDRII and CDRIII regions as shown inFIG. 11 correspond to the V_(L) CDRI, V_(L) CDRII and V_(L) CDRIIIsequences shown in SEQ ID NO.s 35,36 and 37 respectively, and that theamino acid sequences of the CDRI, CDRII and CDRIII regions as shown inFIG. 12 correspond to the V_(L) CDRI, V_(L) CDRII and V_(L) CDRIIIsequences shown in SEQ ID NO.s 25,26 and 27 respectively.

The present invention also provides a process of providing a humanmonoclonal antibody to the TSH receptor substantially as hereinbeforedescribed, which process comprises:

-   -   (i) providing a source of lymphocytes from a subject, which        subject has TSH receptor antibody activity of greater than about        0.04 units of NIBSC 90/672 per mL of serum with respect to        inhibition of TSH binding to the TSH receptor;    -   (ii) isolating lymphocytes from said lymphocyte source of (i);    -   (iii) immortalising the isolated lymphocytes; and    -   (iv) cloning the immortalised lymphocytes so as to produce an        immortalised colony secreting a human monoclonal antibody to the        TSH receptor substantially as hereinbefore described.

Alternatively, a process of providing a human monoclonal antibody to theTSH receptor substantially as hereinbefore described can be defined as aprocess which comprises:

-   -   (i) providing a source of lymphocytes from a subject, which        subject has TSH receptor antibody activity of greater than about        0.1 units of NIBSC 90/672 per mL of serum with respect to        stimulatory activity of cAMP production by cells expressing the        TSH receptor;    -   (ii) isolating lymphocytes from said lymphocyte source of (i);    -   (iii) immortalising the isolated lymphocytes; and    -   (iv) cloning the immortalised lymphocytes so as to produce an        immortalised colony secreting a human monoclonal antibody to the        TSH receptor substantially as hereinbefore described.

Preferably a process according to the present invention comprisesisolating lymphocytes from peripheral blood, thyroid tissue, spleentissue, lymph nodes or bone marrow, most typically from peripheralblood. Typically, the source of lymphocytes for use in a methodaccording to the present invention can be further characterised as beingobtained from a subject having serum TSH receptor antibody levels ofgreater than about 0.1 units of NIBSC 90/672 per mL with respect toinhibition of TSH binding to the TSH receptor, or more typically greaterthan about 0.2 units of NIBSC 90/672 per mL with respect to inhibitionof TSH binding to the TSH receptor, or more typically greater than about0.3 units of NIBSC 90/672 per mL with respect to inhibition of TSHbinding to the TSH receptor and preferably being in the range of about0.3 to 0.5 units of NIBSC 90/672 per mL or greater with respect toinhibition of TSH binding to the TSH receptor. Alternatively, oradditionally, the source of lymphocytes for use in a method according tothe present invention can typically be further characterised as beingobtained from a subject having serum TSH receptor antibody levels ofgreater than about 0.2 units of NIBSC 90/672 per mL with respect tostimulatory activity of cAMP production by cells expressing the TSHreceptor, or more typically greater than about 0.5 units of NIBSC 90/672per mL with respect to stimulatory activity of cAMP production by cellsexpressing the TSH receptor and preferably being in the range of about0.5 to 1.0 units of NIBSC 90/672 per mL or greater with respect tostimulatory activity of cAMP production by cells expressing the TSHreceptor. It will be appreciated from the above that the immune responseto the TSH receptor of a subject from which lymphocytes are isolatedshould preferably be in a highly active phase.

Preferably a process according to the present invention comprisesinfecting the isolated lymphocytes with Epstein Barr virus, and suitablythe thus immortalised lymphocytes are fused with a mouse/human cellline. Suitably a process according to the present invention furthercomprises screening the resulting clones for TSH receptor antibodies,for example by inhibition of ¹²⁵I TSH binding to the TSH receptor in anassay system which has a sensitivity of at least about 1 unit/L of NIBSC90/672.

The present invention further provides a process of preparing a humanrecombinant antibody, or one or more fragments thereof, to the TSHreceptor, which process comprises cloning and expression of a humanmonoclonal antibody to the TSH receptor as provided by the presentinvention by a process substantially as hereinbefore described, or oneor more fragments derived therefrom.

The present invention further provides a human monoclonal or recombinantantibody to the TSH receptor obtained by a process substantially asdescribed above. Preferably such an obtained human monoclonal orrecombinant antibody to the TSH receptor according to the presentinvention, can be characterised by an inhibitory activity with respectto TSH binding to the TSH receptor, of at least about 15 units ofInternational Standard NIBSC 90/672 per mg, more preferably of at leastabout 30 units of International Standard NIBSC 90/672 per mg, morepreferably of at least about 60 units of International Standard NIBSC90/672 per mg, or more preferably of at least about 120 units ofInternational Standard NIBSC 90/672 per mg, or one or more fragments ofsuch a human monoclonal or recombinant antibody.

More particularly, it may be preferred that such a human monoclonal orrecombinant antibody according to the present invention, can be furthercharacterised by a stimulatory activity with respect to cAMP productionby cells expressing the TSH receptor, of at least about 30 units ofInternational Standard NIBSC 90/672 per mg, more preferably of at leastabout 60 units of International Standard NIBSC 90/672 per mg, morepreferably of at least about 120 units of International Standard NIBSC90/672 per mg, or more preferably of at least about 240 units ofInternational Standard NIBSC 90/672 per mg, or one or more fragments ofsuch a human monoclonal or recombinant antibody.

In a preferred embodiment of the present invention, such a humanmonoclonal or recombinant antibody according to the present invention,can be characterised by:

-   -   (i) an inhibitory activity with respect to TSH binding to the        TSH receptor, of at least about 15 units of International        Standard NIBSC 90/672 per mg, more preferably of at least about        30 units of International Standard NIBSC 90/672 per mg, more        preferably of at least about 60 units of International Standard        NIBSC 90/672 per mg, or more preferably of at least about 120        units of International Standard NIBSC 90/672 per mg; and    -   (ii) a stimulatory activity with respect to cAMP production by        cells expressing the TSH receptor, of at least about 30 units of        International Standard NIBSC 90/672 per mg, more preferably of        at least about 60 units of International Standard NIBSC 90/672        per mg, more preferably of at least about 120 units of        International Standard NIBSC 90/672 per mg, or more preferably        of at least about 240 units of International Standard NIBSC        90/672 per mg;        or one or more fragments of such a human monoclonal or        recombinant antibody.

It may also be preferred that one or more fragments of a thus obtainedhuman monoclonal or recombinant antibody according to the presentinvention, in particular for example one or more Fab fragments thereof,can be characterised by an inhibitory activity with respect to TSHbinding to the TSH receptor, of at least about 30 units of InternationalStandard NIBSC 90/672 per mg, more preferably of at least about 60 unitsof International Standard NIBSC 90/672 per mg, more preferably of atleast about 120 units of International Standard NIBSC 90/672 per mg, ormore preferably of at least about 240 units of International StandardNIBSC 90/672 per mg. It may also be preferred that such one or morefragments can be characterised by a stimulatory activity with respect tocAMP production by cells expressing the TSH receptor, of at least about50 units of International Standard NIBSC 90/672 per mg, or morepreferably of at least about 100 units of International Standard NIBSC90/672 per mg, or more preferably of at least about 200 units ofInternational Standard NIBSC 90/672 per mg, or more preferably of atleast about 400 units of International Standard NIBSC 90/672 per mg.

More preferably, such one or more Fab fragments can be characterised by:

-   -   (i) an inhibitory activity with respect to TSH binding to the        TSH receptor, of at least about 30 units of International        Standard NIBSC 90/672 per mg, more preferably of at least about        60 units of International Standard NIBSC 90/672 per mg, more        preferably of at least about 120 units of International Standard        NIBSC 90/672 per mg, or more preferably of at least about 240        units of International Standard NIBSC 90/672 per mg; and    -   (ii) a stimulatory activity with respect to cAMP production by        cells expressing the TSH receptor, of at least about 50 units of        International Standard NIBSC 90/672 per mg, or more preferably        of at least about 100 units of International Standard NIBSC        90/672 per mg, or more preferably of at least about 200 units of        International Standard NIBSC 90/672 per mg, or more preferably        of at least about 400 units of International Standard NIBSC        90/672 per mg.

A process substantially as described above may further comprise afurther process stage whereby the obtained human monoclonal orrecombinant antibody is subjected to suitable further processingtechniques (such as suitable mutagenesis techniques, such as spotmutations or the like), so as to obtain a further binding partner forthe TSH receptor that can compete with a binding partner substantiallyas herein described (such as hMAb TSHR 1) for interaction with the TSHreceptor. Such further processing techniques are well known to one ofordinary skill in the art. The present invention further provides afurther binding partner to the TSH receptor obtained by such furtherprocessing techniques.

Preferably such a further binding partner for the TSH receptor cancomprise a monoclonal or recombinant antibody and can be characterisedby an inhibitory activity with respect to TSH binding to the TSHreceptor, of at least about 15 units of International Standard NIBSC90/672 per mg, more preferably of at least about 30 units ofInternational Standard NIBSC 90/672 per mg, more preferably of at leastabout 60 units of International Standard NIBSC 90/672 per mg, or morepreferably of at least about 120 units of International Standard NIBSC90/672 per mg, or one or more fragments thereof. It may also bepreferred that such a further binding partner according to the presentinvention, can be characterised by a stimulatory activity with respectto cAMP production by cells expressing the TSH receptor, of at leastabout 30 units of International Standard NIBSC 90/672 per mg, morepreferably of at least about 60 units of International Standard NIBSC90/672 per mg, more preferably of at least about 120 units ofInternational Standard NIBSC 90/672 per mg, or more preferably of atleast about 240 units of International Standard NIBSC 90/672 per mg, orone or more fragments thereof.

It may also be even more preferred that such a further binding partnerof the present invention, can be characterised by:

-   -   (i) an inhibitory activity with respect to TSH binding to the        TSH receptor, of at least about 15 units of International        Standard NIBSC 90/672 per mg, more preferably of at least about        30 units of International Standard NIBSC 90/672 per mg, more        preferably of at least about 60 units of International Standard        NIBSC 90/672 per mg, or more preferably of at least about 120        units of International Standard NIBSC 90/672 per mg; and    -   (ii) a stimulatory activity with respect to cAMP production by        cells expressing the TSH receptor, of at least about 30 units of        International Standard NIBSC 90/672 per mg, more preferably of        at least about 60 units of International Standard NIBSC 90/672        per mg, more preferably of at least about 120 units of        International Standard NIBSC 90/672 per mg, or even more        preferably of at least about 240 units of International Standard        NIBSC 90/672 per mg;        or one or more fragments thereof.

A binding partner for the TSH receptor (typically a human monoclonal orrecombinant antibody) according to the present invention may havediagnostic and therapeutic applications.

Accordingly, a binding partner for the TSH receptor (typically a humanmonoclonal or recombinant antibody) according to the present inventioncan be employed in screening methods for detecting autoantibodies to theTSH receptor in patient sera and also in diagnostic methods. In thisway, a binding partner for the TSH receptor (typically a humanmonoclonal or recombinant antibody) according to the present inventioncan be employed in place of, or in addition to, competitors hithertodescribed for use in screening methods for detecting autoantibodies tothe TSH receptor and also in diagnostic methods. Similarly, a bindingpartner for the TSH receptor (typically a human monoclonal orrecombinant antibody) according to the present invention can be employedin place of, or in addition to, competitors hitherto described for usein kits for use in detecting autoantibodies to the TSH receptor.

The present invention also provides, therefore, a method of screeningfor autoantibodies to the TSH receptor in a sample of body fluidobtained from a subject suspected of suffering from, susceptible to,having or recovering from autoimmune disease associated with an immunereaction to a TSH receptor, said method comprising:

-   -   (a) providing said sample of body fluid from said subject;    -   (b) providing one or more pairs of binding molecules, wherein a        first molecule of said binding pair comprises a binding partner        or further binding partner for the TSH receptor (typically a        human monoclonal or recombinant antibody) according to the        present invention and a second molecule of said binding pair        comprises a binding region with which said binding partner or        further binding partner interacts;    -   (c) contacting said sample with said one or more pairs of        binding molecules so as to permit said second molecule of said        binding pair to interact with either (i) autoantibodies to the        TSH receptor present in said sample, or (ii) said binding        partner or further binding partner for the TSH receptor        (typically a human monoclonal or recombinant antibody); and    -   (d) monitoring the interaction of said second molecule of said        binding pair with said autoantibodies present in said sample,        thereby providing an indication of the presence of said        autoantibodies to the TSH receptor in said sample.

A method according to the present invention for the detection ofautoantibodies as described above is particularly advantageous in termsof the level of sensitivity that can be achieved by use thereof. Thiscan be further illustrated by reference to the Examples and Figures,where FIG. 3B shows a graphical representation of a comparison betweenan assay for TSHR autoantibodies based on hMAb TSHR1-biotin and earlierassays. The sensitivity of the assay based on hMAb TSHRI-biotin isclearly superior according to concentration of the internationalstandard NIBSC 90/672 detectable. This was confirmed in a study of serafrom 72 patients with Graves' disease shown in FIG. 3C.

There is further provided by the present invention, therefore, a methodof screening for autoantibodies to the TSH receptor in a sample of bodyfluid obtained from a subject suspected of suffering from, susceptibleto, having or recovering from autoimmune disease associated with animmune reaction to the TSH receptor, said method comprising:

-   -   (a) providing said sample of body fluid from said subject;    -   (b) providing one or more pairs of binding molecules, wherein a        first molecule of said binding pair comprises a binding partner        or further binding partner for the TSH receptor (typically a        human monoclonal or recombinant antibody) according to the        present invention and a second molecule of said binding pair        comprises a binding region with which said binding partner or        further binding partner interacts, wherein the interaction of        said binding molecules is such that an autoantibody titer in        said sample essentially corresponding to 0.4 U/L of        International Standard NIBSC 90/672 is detectable;    -   (c) contacting said sample with said one or more pairs of        binding molecules so as to permit said second molecule of said        binding pair to interact with either (i) autoantibodies to the        TSH receptor present in said sample, or (ii) said binding        partner or further binding partner for the TSH receptor        (typically a human monoclonal or recombinant antibody) according        to the present invention; and    -   (d) monitoring the interaction of said second molecule of said        binding pair with said autoantibodies present in said sample,        thereby providing an indication of the presence of said        autoantibodies to the TSH receptor in said sample.

The above sensitivity can also be achieved in an assay method or kitaccording to the present invention by the use of a human or non-humanpolyclonal antibody to the TSH receptor, TSH or one or more variants,analogs, derivatives or fragments thereof, or a binding partner for theTSH receptor which has an affinity for the TSH receptor of 10¹⁰ molar⁻¹or greater, which generally exhibit a sufficient affinity for the TSHreceptor so that a method or kit of the defined sensitivity is provided.The preparation of such polyclonal antibodies, TSH or one or morevariants, analogs, derivatives or fragments thereof, is well known inthe art. For example, superactive analogs of TSH are described inNature, Biotechnology, Volume 14, October 1995, pages 1257-1263,although this article does not disclose the use of such superactive TSHin a method or kit as is now provided by the present invention.

There is further provided by the present invention, therefore, a methodof screening for autoantibodies to the TSH receptor in a sample of bodyfluid obtained from a subject suspected of suffering from, susceptibleto, having or recovering from autoimmune disease associated with animmune reaction to the TSH receptor, said method comprising:

-   -   (a) providing said sample of body fluid from said subject;    -   (b) providing one or more pairs of binding molecules, wherein a        first molecule of said binding pair comprises a human or        non-human polyclonal antibody to the TSH receptor and a second        molecule of said binding pair comprises a binding region with        which said polyclonal antibody interacts, wherein the        interaction of said binding molecules is such that an        autoantibody titer in said sample essentially corresponding to        0.4 U/L of International Standard NIBSC 90/672 is detectable;    -   (c) contacting said sample with said one or more pairs of        binding molecules so as to permit said second molecule of said        binding pair to interact with either (i) autoantibodies to the        TSH receptor present in said sample, or (ii) said polyclonal        antibody; and    -   (d) monitoring the interaction of said second molecule of said        binding pair with said autoantibodies present in said sample,        thereby providing an indication of the presence of said        autoantibodies to the TSH receptor in said sample.

There is also provided by the present invention a method of screeningfor autoantibodies to the TSH receptor in a sample of body fluidobtained from a subject suspected of suffering from, susceptible to,having or recovering from autoimmune disease associated with an immunereaction to the TSH receptor, said method comprising:

-   -   (a) providing said sample of body fluid from said subject;    -   (b) providing one or more pairs of binding molecules, wherein a        first molecule of said binding pair comprises TSH or one or more        variants, analogs, derivatives or fragments thereof, and a        second molecule of said binding pair comprises a binding region        with which said TSH or one or more variants, analogs,        derivatives or fragments thereof interacts, wherein the        interaction of said binding molecules is such that an        autoantibody titer in said sample essentially corresponding to        0.4 U/L of International Standard NIBSC 90/672 is detectable;    -   (c) contacting said sample with said one or more pairs of        binding molecules so as to permit said second molecule of said        binding pair to interact with either (i) autoantibodies to the        TSH receptor present in said sample, or (ii) said TSH or one or        more variants, analogs, derivatives or fragments thereof; and    -   (d) monitoring the interaction of said second molecule of said        binding pair with said autoantibodies present in said sample,        thereby providing an indication of the presence of said        autoantibodies to the TSH receptor in said sample.

There is also still further provided a method of screening forautoantibodies to the TSH receptor in a sample of body fluid obtainedfrom a subject suspected of suffering from, susceptible to, having orrecovering from autoimmune disease associated with an immune reaction tothe TSH receptor, said method comprising:

-   -   (a) providing said sample of body fluid from said subject;    -   (b) providing one or more pairs of binding molecules, wherein a        first molecule of said binding pair comprises a binding partner        for the TSH receptor which has an affinity for the TSH receptor        of 10¹⁰ molar⁻¹ or greater and a second molecule of said binding        pair comprises a binding region with which said binding partner        interacts, wherein the interaction of said binding molecules is        such that an autoantibody titer in said sample essentially        corresponding to 0.4 U/L of International Standard NIBSC 90/672        is detectable;    -   (c) contacting said sample with said one or more pairs of        binding molecules so as to permit said second molecule of said        binding pair to interact with either (i) autoantibodies to the        TSH receptor present in said sample, or (ii) said binding        partner for the TSH; and    -   (d) monitoring the interaction of said second molecule of said        binding pair with said autoantibodies present in said sample,        thereby providing an indication of the presence of said        autoantibodies to the TSH receptor in said sample.

There is also provided by the present invention use of a binding partneror further binding partner for the TSH receptor (typically a humanmonoclonal or recombinant antibody) according to the present invention,for detecting autoantibodies to the TSH receptor in a sample of bodyfluid obtained from a subject suspected of suffering from, susceptibleto, having or recovering from autoimmune disease associated with animmune reaction to the TSH receptor, wherein the interaction of saidbinding partner or further binding partner with the TSH receptor is suchthat an autoantibody titer in said sample essentially corresponding to0.4 U/L of International Standard NIBSC 90/672 is detectable.

There is also provided use of a human or non-human polyclonal antibodyto the TSH receptor, for detecting autoantibodies to the TSH receptor ina sample of body fluid obtained from a subject suspected of sufferingfrom, susceptible to, having or recovering from autoimmune diseaseassociated with an immune reaction to the TSH receptor, wherein theinteraction of said polyclonal antibody with the TSH receptor is suchthat an autoantibody titer in said sample essentially corresponding to0.4 U/L of International Standard NIBSC 90/672 is detectable.

There is also provided use of TSH or one or more variants, analogs,derivatives or fragments thereof, for detecting autoantibodies to theTSH receptor in a sample of body fluid obtained from a subject suspectedof suffering from, susceptible to, having or recovering from autoimmunedisease associated with an immune reaction to the TSH receptor, whereinthe interaction of said TSH or one or more variants, analogs,derivatives or fragments thereof with the TSH receptor is such that anautoantibody titer in said sample essentially corresponding to 0.4 U/Lof International Standard NIBSC 90/672 is detectable.

There is still further provided use of a binding partner for the TSHreceptor which has an affinity for the TSH receptor of 10¹⁰ molar⁻¹ orgreater, for detecting autoantibodies to the TSH receptor in a sample ofbody fluid obtained from a subject suspected of suffering from,susceptible to, having or recovering from autoimmune disease associatedwith an immune reaction to the TSH receptor, wherein the interaction ofsaid binding partner with the TSH receptor is such that an autoantibodytiter in said sample essentially corresponding to 0.4 U/L ofInternational Standard NIBSC 90/672 is detectable.

It will be appreciated that binding molecules of the one or more bindingpairs can be antigen-antibody (for example, [TSH receptor orepitope]-[monoclonal or recombinant TSH receptor antibody]),anti-idiotypic antibody-monoclonal or recombinant TSH receptor antibodyor novel TSH receptor antibody binding member-monoclonal or recombinantTSH receptor antibody. Preferably, the binding molecules of the bindingpairs are antigen-antibody, namely, [TSH receptor or one or moreepitopes thereof 1-[monoclonal or recombinant TSH receptor antibody],where the epitopes may be “free standing” or present in a largerscaffold polypeptide or the like.

Preferably, the present invention provides a method of screening forautoantibodies to the TSH receptor in a sample of body fluid obtainedfrom a subject suspected of suffering from, susceptible to, having orrecovering from autoimmune disease associated with an immune reaction toa TSH receptor, said method comprising:

-   -   (a) providing said sample of body fluid from said subject;    -   (b) contacting said sample with (i) a full length TSH receptor,        or one or more epitopes thereof or a polypeptide comprising one        or more epitopes of a TSH receptor, and (ii) a binding partner        or further binding partner for the TSH receptor (typically a        human monoclonal or recombinant antibody) according to the        present invention, under conditions that allow interaction of        the TSH receptor with autoantibodies produced in response to the        TSH receptor, so as to permit said TSH receptor, or said one or        more epitopes thereof or said polypeptide, to interact with        either autoantibodies to the TSH receptor present in said        sample, or said binding partner or further binding partner for        the TSH receptor (typically a human monoclonal or recombinant        antibody); and    -   (c) monitoring the interaction of said TSH receptor, or said one        or more epitopes thereof or said polypeptide, with said        autoantibodies present in said sample, thereby providing an        indication of the presence of said autoantibodies to the TSH        receptor in said sample.

In certain embodiments, a method according to the present invention mayalso employ one or more competitors that compete in the interaction of apolyclonal antibody, TSH or one or more variants, analogs, derivativesor fragments thereof, or a binding partner or further binding partnerfor the TSH receptor substantially as described above in the specificembodiments of methods as provided by the present invention and thesecond molecule of the binding pair, or the TSH receptor, or the one ormore epitopes thereof or the polypeptide. Such competitors may compriseTSH, or one or more monoclonals reactive with the TSH receptor, such asmouse monoclonals reactive with the TSH receptor.

Preferably, a method according to the present invention as referred toabove, further comprises providing labelling means for a binding partneror further binding partner for the TSH receptor (typically a humanmonoclonal or recombinant antibody) according to the present inventionand where appropriate one or more competitors as described above,suitable labelling means including enzymic labels, isotopic labels,chemiluminescent labels, fluorescent labels, dyes and the like.

The present invention also provides, a kit for screening forautoantibodies to the TSH receptor in a sample of body fluid obtainedfrom a subject suspected of suffering from, susceptible to, having orrecovering from autoimmune disease associated with an immune reaction toa TSH receptor, said kit comprising:

-   -   (a) one or more pairs of binding molecules, wherein a first        molecule of said binding pair comprises a binding partner or        further binding partner for the TSH receptor (typically a human        monoclonal or recombinant antibody) according to the present        invention and a second molecule of said binding pair comprises a        binding region with which said binding partner or further        binding partner interacts;    -   (b) means for contacting said sample of body fluid from said        subject with said one or more pairs of binding molecules so as        to permit said second molecule of said binding pair to interact        with either (i) autoantibodies to the TSH receptor present in        said sample, or (ii) said binding partner or further binding        partner for the TSH receptor (typically a human monoclonal or        recombinant antibody); and    -   (c) means for monitoring the interaction of said second molecule        of said binding pair with said autoantibodies present in said        sample, thereby providing an indication of the presence of said        autoantibodies to the TSH receptor in said sample.

The present invention also provides a kit for screening forautoantibodies to the TSH receptor in a sample of body fluid obtainedfrom a subject suspected of suffering from, susceptible to, having orrecovering from autoimmune disease associated with an immune reaction tothe TSH receptor, said kit comprising:

-   -   (a) one or more pairs of binding molecules, wherein a first        molecule of said binding pair comprises a binding partner or        further binding partner for the TSH receptor (typically a human        monoclonal or recombinant antibody) according to the, present        invention and a second molecule of said binding pair comprises a        binding region with which said binding partner or further        binding partner interacts, wherein the interaction of said        binding molecules is such that an autoantibody titer in said        sample essentially corresponding to 0.4 U/L of International        Standard NIBSC 90/672 is detectable;    -   (b) means for contacting said sample of body fluid from said        subject with said one or more pairs of binding molecules so as        to permit said second molecule of said binding pair to interact        with either (i) autoantibodies to the TSH receptor present in        said sample, or (ii) said binding partner or further binding        partner for the TSH receptor (typically a human monoclonal or        recombinant antibody) according to the present invention; and    -   (c) means for monitoring the interaction of said second molecule        of said binding pair with said autoantibodies present in said        sample, thereby providing an indication of the presence of said        autoantibodies to the TSH receptor in said sample.

There is also provided a kit for screening for autoantibodies to the TSHreceptor in a sample of body fluid obtained from a subject suspected ofsuffering from, susceptible to, having or recovering from autoimmunedisease associated with an immune reaction to the TSH receptor, said kitcomprising:

-   -   (a) one or more pairs of binding molecules, wherein a first        molecule of said binding pair comprises a human or non-human        polyclonal antibody to the TSH receptor and a second molecule of        said binding pair comprises a binding region with which said        polyclonal antibody interacts, wherein the interaction of said        binding molecules is such that an autoantibody titer in said        sample essentially corresponding to 0.4 U/L of International        Standard NIBSC 90/672 is detectable;    -   (b) means for contacting said sample of body fluid from said        subject with said one or more pairs of binding molecules so as        to permit said second molecule of said binding pair to interact        with either (i) autoantibodies to the TSH receptor present, in        said sample, or (ii) said polyclonal antibody; and    -   (c) means for monitoring the interaction of said second molecule        of said binding pair with said autoantibodies present in said        sample, thereby providing an indication of the presence of said        autoantibodies to the TSH receptor in said sample.

There is also provided a kit for screening for autoantibodies to the TSHreceptor in a sample of body fluid obtained from a subject suspected ofsuffering from, susceptible to, having or recovering from autoimmunedisease associated with an immune reaction to the TSH receptor, said kitcomprising:

-   -   (a) one or more pairs of binding molecules, wherein a first        molecule of said binding pair comprises TSH or one or more        variants, analogs, derivatives or fragments thereof, and a        second molecule of said binding pair comprises a binding region        with which said TSH or one or more variants, analogs,        derivatives or fragments thereof interacts, wherein the        interaction of said binding molecules is such that an        autoantibody titer in said sample essentially corresponding to        0.4 U/L of International Standard NIBSC 90/672 is detectable;    -   (b) means for contacting said sample of body fluid from said        subject with said one or more pairs of binding molecules so as        to permit said second molecule of said binding pair to interact        with either (i) autoantibodies to the TSH receptor present in        said sample, or (ii) TSH or one or more variants, analogs,        derivatives or fragments thereof; and    -   (c) means for monitoring the interaction of said second molecule        of said binding pair with said autoantibodies present in said        sample, thereby providing an indication of the presence of said        autoantibodies to the TSH receptor in said sample.

There is also provided a kit for screening for autoantibodies to the TSHreceptor in a sample of body fluid obtained from a subject suspected ofsuffering from, susceptible to, having or recovering from autoimmunedisease associated with an immune reaction to the TSH receptor, said kitcomprising:

-   -   (a) one or more pairs of binding molecules, wherein a first        molecule of said binding pair comprises a binding partner for        the TSH receptor which has an affinity for the TSH receptor of        10¹⁰ molar⁻¹ or greater and a second molecule of said binding        pair comprises a binding region with which said binding partner        interacts, wherein the interaction of said binding molecules is        such that an autoantibody titer in said sample essentially        corresponding to 0.4 U/L of International Standard NIBSC 90/672        is detectable;    -   (b) means for contacting said sample of body fluid from said        subject with said one or more pairs of binding molecules so as        to permit said second molecule of said binding pair to interact        with either (i) autoantibodies to the TSH receptor present in        said sample, or (ii) said binding partner for the TSH receptor;        and    -   (c) means for monitoring the interaction of said second molecule        of said binding pair with said autoantibodies present in said        sample, thereby providing an indication of the presence of said        autoantibodies to the TSH receptor in said sample.

It will be appreciated that binding molecules of the one or more bindingpairs can be antigen-antibody (for example, [TSH receptor orepitope]-[monoclonal or recombinant TSH receptor antibody]),anti-idiotypic antibody-monoclonal or recombinant TSH receptor antibodyor novel TSH receptor antibody binding member-monoclonal or recombinantTSH receptor antibody. Preferably, the binding molecules of the bindingpairs are antigen-antibody, namely, [TSH receptor or one or moreepitopes thereof]-[monoclonal or recombinant TSH receptor antibody],where the epitopes may be “free standing” or present in a largerscaffold polypeptide or the like.

The present invention preferably provides a kit for screening forautoantibodies to the TSH receptor in a sample of body fluid obtainedfrom a subject suspected of suffering from, susceptible to, having orrecovering from autoimmune disease associated with an immune reaction tothe TSH receptor, said kit comprising:

-   -   (a) a full length TSH receptor, or one or more epitopes thereof        or a polypeptide comprising one or more epitopes of the TSH        receptor;    -   (b) a binding partner or further binding partner for the TSH        receptor (typically a human monoclonal or recombinant antibody)        according to the present invention;    -   (c) means for contacting said sample of body fluid from said        subject, said TSH receptor, or said one or more epitopes thereof        or said polypeptide, and said binding partner or further binding        partner for the TSH receptor (typically a human monoclonal or        recombinant antibody), under conditions that allow interaction        of the TSH receptor with autoantibodies produced in response to        the TSH receptor, so as to permit said TSH receptor, or said one        or more epitopes thereof or said polypeptide, to interact with        either autoantibodies to a TSH receptor present in said sample,        or said binding partner or further binding partner for the TSH        receptor (typically a human monoclonal or recombinant antibody);        and    -   (d) means for monitoring the interaction of said TSH receptor,        or said one or more epitopes thereof or said polypeptide, with        said autoantibodies present in said sample, thereby providing an        indication of the presence of said autoantibodies to the TSH        receptor in said sample.

In certain embodiments, a kit according to the present invention mayfurther comprise one or more competitors that compete in the interactionof a polyclonal antibody, TSH or one or more variants, analogs,derivatives or fragments thereof, or a binding partner or furtherbinding partner for the TSH receptor, as respectively defined above, andthe second molecule of the binding pair, or the TSH receptor, or the oneor more epitopes thereof or the polypeptide. Such competitors maycomprise TSH, or one or more monoclonals reactive with the TSH receptor,such as mouse monoclonals reactive with the TSH receptor.

Suitably, a kit as referred to above further comprises labelling meansfor a binding partner or further binding partner for the TSH receptor(typically a human monoclonal or recombinant antibody) according to thepresent invention and where appropriate one or more competitors asdescribed above, suitable labelling means being substantially ashereinbefore described.

In the presence of autoantibodies to the TSH receptor, binding of theTSH receptor to a binding partner for the TSH receptor (typically ahuman monoclonal or recombinant antibody) in a method or kit asdescribed above will be decreased.

A binding partner or further binding partner for the TSH receptor(typically a human monoclonal or recombinant antibody) according to thepresent invention can also be employed in assay methods and kitssubstantially as described above for TSH and related ligands.

The present invention also provides, therefore, a method of assaying TSHand related ligands, said method comprising:

-   -   (a) providing a sample suspected of containing or containing TSH        or related ligands;    -   (b) providing one or more pairs of binding molecules, wherein a        first molecule of said binding pair comprises a binding partner        or further binding partner for the TSH receptor (typically a        human monoclonal or recombinant antibody) according to the        present invention and a second molecule of said binding pair        comprises a binding region with which said binding partner or        further binding partner interacts;    -   (c) contacting said sample with said one or more pairs of        binding molecules so as to permit said second molecule of said        binding pair to interact with either (i) TSH or related ligands        present in said sample, or (ii) said binding partner or further        binding partner for the TSH receptor (typically a human        monoclonal or recombinant antibody); and    -   (d) monitoring the interaction of said second molecule of said        binding pair with TSH or related ligands present in said sample,        thereby providing an indication of the presence of TSH or        related ligands in said sample.

The present invention also provides a kit for assaying TSH or relatedligands, said kit comprising:

-   -   (a) one or more pairs of binding molecules, wherein a first        molecule of said binding pair comprises a binding partner or        further binding partner for the TSH receptor (typically a human        monoclonal or recombinant antibody) according to the present        invention and a second molecule of said binding pair comprises a        binding region with which said binding partner or further        binding partner interacts;    -   (b) means for contacting a sample suspected of containing or        containing TSH or related ligands with said one or more pairs of        binding molecules so as to permit said second molecule of said        binding pair to interact with either (i) TSH or related ligands        present in said sample, or (ii) said binding partner or further        binding partner for the TSH receptor (typically a human        monoclonal or recombinant antibody); and    -   (c) means for monitoring the interaction of said second molecule        of said binding pair with TSH or related ligands present in said        sample, thereby providing an indication of the presence of TSH        or related ligands in said sample.

The present invention also further provides a method of identifying afurther binding partner for the TSH receptor, which further bindingpartner is capable of binding to the TSH receptor and which competes forbinding to the TSH receptor with a binding partner for the TSH receptorsubstantially as hereinbefore described, which further binding partnerdoes not comprise TSH, which method comprises:

-   -   (a) providing one or more pairs of binding molecules, wherein a        first molecule of said binding pair comprises a binding partner        for the TSH receptor substantially as hereinbefore described and        a second molecule of said binding pair comprises a binding        region with which said binding partner interacts;    -   (b) providing a further binding molecule to be assayed as a        potential further binding partner for the TSH receptor which        competes for binding to the TSH receptor with said first        molecule of said binding pair of (a);    -   (c) contacting said further binding molecule of (b) with said        one or more pairs of binding molecules of (a) so as to permit        said second molecule of said binding pair of (a) to interact        with either (i) said further binding molecule of (b), or (ii)        said first molecule of said binding pair of (a); and    -   (d) monitoring the interaction of said second molecule of said        binding pair of (a) with said further binding molecule of (b),        and thereby assessing whether said further binding molecule        of (b) competes for binding to the TSH receptor with said first        molecule of said binding pair of (a).

The present invention also provides a kit for identifying a furtherbinding partner for the TSH receptor, which further binding partner iscapable of binding to the TSH receptor and which competes for binding tothe TSH receptor with a binding partner for the TSH receptorsubstantially as hereinbefore described, which further binding partnerdoes not comprise TSH, which kit comprises:

-   -   (a) one or more pairs of binding molecules, wherein a first        molecule of said binding pair comprises a binding partner for        the TSH receptor substantially as hereinbefore described and a        second molecule of said binding pair comprises a binding region        with which said binding partner interacts;    -   (b) means for contacting said one or more pairs of binding        molecules of (a) with a further binding molecule to be assayed        as a potential further binding partner for the TSH receptor        which competes for binding to the TSH receptor with said first        molecule of said binding pair of (a), so as to permit said        second molecule of said binding pair of (a) to interact with        either (i) said further binding molecule, or (ii) said first        molecule of said binding pair of (a); and    -   (c) means for monitoring the interaction of said second molecule        of said binding pair of (a) with said further binding molecule,        and thereby assessing whether said further binding molecule        competes for binding to the TSH receptor with said first        molecule of said binding pair of (a).

A further application of a binding partner or further binding partnerfor the TSH receptor (typically a human monoclonal or recombinantantibody) according to the present invention is its use to identify andprovide new types of TSH receptor antibody binding sites. There isfurther provided by the present invention, therefore, a process ofidentifying one or more epitope regions of the TSH receptor, whichprocess comprises contacting a binding partner or further bindingpartner for the TSH receptor (typically a human monoclonal orrecombinant antibody) substantially as hereinbefore described with afull length TSH receptor, or one or more fragments thereof, so as toallow interaction of said binding partner or further binding partner forthe TSH receptor with said full length TSH receptor, or said one or morefragments thereof, and identifying the amino acids of said full lengthTSH receptor, or said one or more fragments thereof, with which saidbinding partner or further binding partner interacts. Suitably,interaction of the binding partner or further binding partner withselected fragments of the TSH receptor and the full length TSH receptor,is analysed, so as to identify the amino acids of the TSH receptor withwhich the binding partner interacts.

Furthermore, the present invention allows for generation of antibodiesto the regions of a monoclonal TSH receptor antibody according to thepresent invention which bind the TSH receptor. Such anti-idiotypicantibodies produced in this way could have potential as new ligands forassays of TSH receptor autoantibodies, TSH and related compounds. Alsothey may be effective agents in vivo for regulating the action of TSHreceptor autoantibodies, TSH and related compounds. The presentinvention further provides, therefore, one or more anti-idiotypicantibodies generated to binding regions of a binding partner or furtherbinding partner for the TSH receptor (typically a human monoclonal orrecombinant antibody) substantially as hereinbefore described, and thepreparation thereof is further described by the Examples.

Other methods of identifying and providing new types of antibody bindingsites using monoclonal antibodies are well known. For example byantibody screening of phage-displayed random peptide libraries asdescribed by J C Scott and G P Smith; “Searching for peptide ligandswith an epitope library”; Science 1990; 249 (4967): 386-390 and M AMyers, J M Davies, J C Tong, J Whisstock, M Scealy, I R MacKay, M JRowley; “Conformational epitopes on the diabetes autoantigen GAD₆₅identified by peptide phage display and molecular modelling”; Journal ofImmunology 2000; 165: 3830-3838. Antibody screening of non-peptidecompounds and libraries of non-peptide compounds can also be carriedout.

New types of TSH receptor antibody binding sites identified and providedusing these procedures may also be useful as new ligands in assays forTSH receptor autoantibodies, TSH and related compounds. Furthermore theymay be effective agents in vivo for regulating the action of TSHreceptor autoantibodies, TSH and related compounds.

A binding partner for the TSH receptor or further binding partner(typically a human monoclonal or recombinant antibody) according to thepresent invention substantially as hereinbefore described can also beusefully employed in therapy. There is, therefore, further provided bythe present invention methods of treatment comprising administration ofa binding partner or further binding partner for the TSH receptor(typically a human monoclonal or recombinant antibody) substantially ashereinbefore described, pharmaceutical compositions comprising a bindingpartner or further binding partner for the TSH receptor (typically ahuman monoclonal or recombinant antibody) substantially as hereinbeforedescribed (together with one or more pharmaceutically acceptablecarriers, diluents or excipients therefor), and use of a binding partneror further binding partner for the TSH receptor (typically a humanmonoclonal or recombinant antibody) substantially as hereinbeforedescribed in the manufacture of a medicament or composition.

A binding partner for the TSH receptor, in particular a human monoclonalantibody to the TSH receptor derived from patients' lymphocytesaccording to the present invention, is a valuable reagent forunderstanding the pathogenesis of Graves' disease and for developing newmethods of measuring TSH receptor autoantibodies, for example asreplacements for TSH in competitive binding assays substantially ashereinbefore described. Also, a stimulating binding partner according tothe present invention has in vivo applications when tissue containingthe TSH receptor (eg thyroid tissue or thyroid cancer tissue) requiresstimulation. The present invention provides, therefore, a medicament orcomposition for use in stimulating thyroid tissue, and/or tissuecontaining the TSH receptor. In particular, a stimulating bindingpartner for the TSH receptor (typically a human monoclonal orrecombinant antibody) according to the present invention can be employedin oncology, and in particular for use in the diagnosis, management andtreatment of thyroid cancer.

Alternatively, a binding partner or further binding partner for the TSHreceptor according to the present invention can be a powerful TSH orautoantibody antagonist (blocking antibody) and such a blocking TSHreceptor antibody according to the present invention is valuable for invivo applications when the activity of tissue containing the TSHreceptor (eg thyroid tissue or thyroid cancer tissue) requiresinactivation or to be made unresponsive to TSH, TSH receptorautoantibodies or other stimulators.

There is also provided in combination, a binding partner or furtherbinding partner for the TSH receptor substantially as hereinbeforedescribed, together with one or more further agents capable ofinactivating or rendering unresponsive, tissue containing a TSHreceptor, to TSH, TSH receptor autoantibodies or other stimulators.Typically, the one or more further agents act independently of the TSHreceptor.

A particular therapeutic application where TSH receptor autoantibodybinding requires inactivation or inhibition is in the treatment ofdisease of the retro orbital tissues of the eye associated withautoimmunity to the TSH receptor, and the use of a blocking antibodywhich interacts with the TSH receptor, such as 9D33, so as to inhibitTSH receptor autoantibody binding, thus has important therapeuticutility in the treatment of such disease. Treatment of autoimmunedisease which requires inhibition of TSH receptor autoantibody binding,such as the above discussed disease of the retro orbital tissues of theeye associated with autoimmunity to the TSH receptor, may alternativelyemploy an anti-idiotypic antibody to a binding partner or furtherbinding partner as provided by the present invention, and suchanti-idiotypic antibodies form a further aspect of the present inventionas described herein and further preparatory details thereof are providedby the Examples.

More specifically, therefore, the present invention provides use in thetreatment of disease of the retro orbital tissues of the eye associatedwith autoimmunity to the TSH receptor, of a further binding partner tothe TSH receptor, which further binding partner substantially inhibitsbinding to the TSH receptor of a binding partner for the TSH receptor(typically a human monoclonal or recombinant antibody) substantially ashereinbefore described. The present invention further provides use inthe manufacture of a medicament for the treatment of disease of theretro orbital tissues of the eye associated with activation and/orstimulation of the TSH receptor, of a further binding partner to the TSHreceptor, which further binding partner substantially inhibits bindingto the TSH receptor of a binding partner for the TSH receptor (typicallya human monoclonal or recombinant antibody) substantially ashereinbefore described. There is also provided a method of treatingdisease of the retro orbital tissues of the eye associated withautoimmunity to the TSH receptor, which method comprises administrationto a patient suffering from or susceptible to such disease atherapeutically effective amount of a further binding partner to the TSHreceptor, which further binding partner substantially inhibits bindingto the TSH receptor of a binding partner for the TSH receptor (typicallya human monoclonal or recombinant antibody) substantially ashereinbefore described. A further binding partner for use in theseembodiments of the present invention preferably comprises a blockingantibody which can substantially inhibit binding of a binding partner asprovided by the present invention, and as such TSH receptor autoantibodybinding, to the TSH receptor, and a preferred such antibody can comprise9D33 as described herein.

The present invention also provides use of an anti-idiotypic antibodygenerated to a binding region of a binding partner or further bindingpartner according to the present invention, in the treatment of diseaseof the retro orbital tissues of the eye associated with autoimmunity tothe TSH receptor. The present invention further provides use of ananti-idiotypic antibody generated to a binding region of a bindingpartner or further binding partner according to the present invention,in the manufacture of a medicament for the treatment of disease of theretro orbital tissues of the eye associated with activation and/orstimulation of the TSH receptor. There is also provided a method oftreating disease of the retro orbital tissues of the eye associated withautoimmunity to the TSH receptor, which method comprises administrationto a patient suffering from or susceptible to such disease atherapeutically effective amount of an anti-idiotypic antibody generatedto a binding region of a binding partner or further binding partneraccording to the present invention.

One of the major advantages of a monoclonal antibody as provided by thepresent invention over TSH in such in vitro and/or in vivo applicationsis the relative ease with which such antibodies can be manipulated. Forexample, manipulation of the TSH receptor binding region of a monoclonalantibody according to the present invention so as to change thecharacteristics thereof, such as affinity and biologicalcharacteristics, including the degree of TSH agonist or antagonistactivities. Also monoclonal antibodies according to the presentinvention have a much longer half life than TSH in vivo and this mayhave considerable advantages in in vivo applications. Furthermore, thehalf life of the antibodies can be manipulated, for example antibody Fabfragments have a much shorter half life than intact IgG.

Pharmaceutical compositions according to the present invention includethose suitable for oral, parenteral and topical administration, althoughthe most suitable route will generally depend upon the condition of apatient and the specific disease being treated. The precise amount of abinding partner or further binding partner for the TSH receptor(typically a human monoclonal or recombinant antibody) substantially ashereinbefore described to be administered to a patient will be theresponsibility of an attendant physician, although the dose employedwill depend upon a number of factors, including the age and sex of thepatient, the specific disease being treated and the route ofadministration substantially as described above.

There is further provided by the present invention a method ofstimulating thyroid tissue, and/or tissue containing a TSH receptor,which method comprises administering to a patient in need of suchstimulation a diagnostically or therapeutically effective, amount of abinding partner or further binding partner for the TSH receptor(typically a human monoclonal or recombinant antibody) substantially ashereinbefore described.

The present invention also provides in combination, a binding partnerfor the TSH receptor (typically a human monoclonal or recombinantantibody) substantially as hereinbefore described, together with one ormore further agents capable of stimulating thyroid tissue, and/or tissuecontaining a TSH receptor, for simultaneous, separate or sequential usein stimulating thyroid tissue, and/or tissue containing a TSH receptor.Preferably the one or more further agents comprise recombinant human TSHand/or one or more variants, analogs, derivatives or fragments thereof,or variants, analogs or derivatives of such fragments. Alternatively,the one or more further agents can act independently of binding to theTSH receptor.

A binding partner for the TSH receptor or further binding partner(typically a human monoclonal or recombinant antibody) according to thepresent invention can also be employed as a replacement source forpatient serum required to contain TSH receptor antibody or antibodiesfor use in commercial kits. Furthermore, a binding partner or furtherbinding partner for the TSH receptor (typically a human monoclonal orrecombinant antibody) can be provided according to the present inventionin a preparation required to comprise a defined concentration of TSHreceptor antibody or antibodies, and in this way there can be provided apreparation with a defined activity, such as stimulatory activity, withrespect to the TSH receptor. Optionally, such a preparation may furthercomprise one or more further human monoclonal antibodies, such asmonoclonal antibodies to GAD, TPO or the like.

The following illustrative explanations are provided to facilitateunderstanding of certain terms used herein. The explanations areprovided as a convenience and are not limitative of the invention

BINDING PARTNER FOR A TSH RECEPTOR, describes a molecule having abinding specificity for the TSH receptor. A binding partner as describedherein may be naturally derived or wholly or partially syntheticallyproduced. Such a binding partner has a domain or region whichspecifically binds to and is therefore complementary to one or moreepitope regions of the TSH receptor. In particular, a binding partner asdescribed herein can be a monoclonal or recombinant antibody to the TSHreceptor, and more particularly can be a human monoclonal or recombinantantibody to the TSH receptor.

C DOMAIN denotes a region of relatively constant amino acid sequence inantibody molecules.

CDR denotes complementarity determining regions which are present onboth heavy and light chains of antibody molecules and represent regionsof most sequence variability. CDRs represent approximately 15 to 20% ofvariable domains and represent antigen binding sites of an antibody.

FR denotes framework regions and represent the remainder of the variablelight domains and variable heavy domains not present in CDRs.

HC denotes part of a heavy chain of an antibody molecule comprising theheavy chain variable domain and the first domain of an IgG constantregion.

HOST CELL is a cell which has been transformed or transfected, or iscapable of transformation or transfection by an exogenous polynucleotidesequence.

IDENTITY, as known in the art, is the relationship between two or morepolypeptide sequences, or two or more polynucleotide sequences, asdetermined by comparing the sequences.

LC denotes a light chain of an antibody molecule.

NIBSC 90/672 is, an International Standard for thyroid stimulatingantibody. The International Standard for thyroid stimulating activityconsists of a batch of ampoules containing freeze dried plasma proteinsfrom a single human patient with high TSH receptor autoantibodies. Thepreparation has been evaluated in an international collaborative studyand shown to possess both thyroid stimulating and thyroid receptorbinding activity. At the 46^(th) meeting in 1995, the Expert Committeeon Biological Standardization of WHO established the preparation coded90/672 as the International Standard for thyroid stimulating antibody.Each ampoule contains freeze-dried residue of 1.0 ml of a solutioncontaining 0.02M phosphate buffer, dialysed human plasma proteins and0.1 International Units (100 milli-International Units) per ampoule bydefinition.

STIMULATION OF A TSH RECEPTOR by a human monoclonal antibody asdescribed herein denotes the ability thereof to bind to a TSH receptorand to thereby effect, for example, production of cyclic AMP as a resultof such binding to the TSH receptor. Such stimulation is analogous tothe responses seen on binding of TSH, or TSH receptor autoantibodies, tothe TSH receptor and in this way a human monoclonal antibody asdescribed herein essentially provides the same or similar bindingresponses as seen with TSH, or TSH receptor autoantibody, binding to aTSH receptor.

V DOMAIN denotes a region of highly variable amino acid sequence inantibody molecules.

V_(H) DOMAIN denotes variable regions or domains in heavy chains ofantibody molecules.

V_(L) DOMAIN denotes variable regions or domains in light chains ofantibody molecules.

The present invention will now be illustrated by the following Figuresand Examples, which do not limit the scope of the invention in any way.

EXAMPLES Materials & Methods

Lymphocyte Isolation and Clonina of Human Monoclonal TSH ReceptorAutoantibodies

Blood was obtained from a patient with Graves' disease and Type 1diabetes mellitus who had high levels of serum autoantibodies to the TSHreceptor (TRAb). Ethical Committee approval was obtained for thestudies. Peripheral blood lymphocytes were isolated on Ficoll-Paque(Amersham Biosciences; Chalfont St Giles, HPS 4SP, UK) from a 20 mLblood sample and then infected with Epstein Barr virus (EBV) (EuropeanCollection of Cell Cultures-ECACC; Porton Down, SP4 OJG, UK) andcultured on mouse macrophage feeder layers as described before (NHayakawa, L D K E Premawardhana, M Powell, M Masuda, C Arnold, JSanders, M Evans, S Chen, J C Jaume, S Baekkeskov, B Rees Smith, JFurmaniak; “Isolation and characterization of human monoclonalautoantibodies to glutamic acid decarboxylase”; Autoimmunity 2002; 35:343-355). EBV immortalised B lymphocytes were then fused with themouse/human hybrid cell line K6H6/B5 (WL Carroll, K Thilemans, J Dilley,R Levy; “Mouse×human heterohybridomas as fusion partners with human Bcell tumors”; Journal of Immunological Methods 1986; 89: 61-72) andcloned two times by limiting dilution at 5 cells/well and a final timeat ½ cell/well to obtain a single colony (B J Bolton, N K Spurr.“B-lymphocytes” In: R I Freshney, M G Freshney (eds). Culture ofimmortalized cells. Wiley-Liss, New York 1996; 283-297). The originalwells and subsequent clones were screened for TSH receptor autoantibodyby inhibition of ¹²⁵I-TSH binding to solubilised TSH receptor (seebelow). The single clones producing TSH receptor autoantibodies weregrown up in tissue culture flasks.

Production, Purification and Labelling of Monoclonal Tsh ReceptorAntibody Preparations

Mouse TSH receptor MAbs were produced as described before (Y Oda, JSanders, M Evans, A Kiddie, A Munkley, C James, T Richards, J Wills, JFurmaniak, B Rees Smith; “Epitope analysis of the human thyrotropin(TSH) receptor using monoclonal antibodies”; Thyroid 2000; 10:1051-1059) and were also prepared from mice immunised with full lengthTSHR cDNA cloned in pcDNA3.1 (U A Hasan, A M Abai, D R Harper, B W Wren,W J W Morrow; “Nucleic acid immunization: Concepts and techniquesassociated with third generation vaccines”; Journal of ImmunologicalMethods 1999; 229: 1-22).

IgGs were purified from tissue culture supernatants using affinitychromatography on Prosep A (Millipore UK Ltd.; Watford, WD18 SYH, UK)according to the manufacturer's instructions and purity assessed bySDS-polyacrylamide gel electrophoresis (PAGE).

Human heavy chain isotype was determined using a radial diffusion assay(The Binding Site; Birmingham, B29 6AT, UK). Human light chain isotypewas determined using Western blotting with anti-human kappa chain andanti human lambda chain specific mouse monoclonal antibodies(Sigma-Aldrich Company Ltd; Gillingham, SP8 4XT, UK).

The purified IgG preparations were treated with mercuripapain(Sigma-Aldrich) at an enzyme/protein ratio of between 1:10 and 1:100(depending on the particular monoclonal antibody) and passed through aProsep A column to remove any intact IgG or Fc fragment from the Fabpreparation (Y Oda, J Sanders, S Roberts, M Maruyama, R Kato, M Perez, VB Petersen, N Wedlock, J Furmaniak, B Rees Smith; “Bindingcharacteristics of antibodies to the TSH receptor”; Journal of MolecularEndocrinology 1998; 20: 233-244). Intact IgG was undetectable bySDS-PAGE in the Fab preparations. IgG and Fab preparations of themonoclonal antibodies were labelled with ¹²⁵I as described previously (YOda, J Sanders, S Roberts, M Maruyama, R Kato, M Perez, V B Petersen, NWedlock, J Furmaniak, B Rees Smith; “Binding characteristics ofantibodies to the TSH receptor”; Journal of Molecular Endocrinology;1998; 20: 233-244). IgG preparations were labelled with biotin hydrazide(Pierce Rockford IL61105 USA) according to the manufacturersinstructions. Crystals of Fab fragments of the human monoclonal TSHreceptor autoantibody were obtained and their crystal structuredetermined using standard techniques.

Patients

Sera from patients with Graves' disease of different disease durationwere studied. The patients' sera studied showed inhibition of¹²⁵I-labelled TSH binding to the TSH receptor (see below). In addition,sera from 2 patients with Addison's disease (A1 and A2) and high levelsof autoantibodies to 21-OH (113 and 1970 units per mL, RSR kit) and serafrom 2 patients with type 1 diabetes mellitus (D1 and D2) with highlevels of GAD₆₅ (3700 and 37.5 units per mL; RSR kit) were studied.Informed consent for the study was obtained from the patients. Sera fromhealthy blood donors (purchased from Golden West Biologicals, Vista,Calif. 92083, USA) were also studied. TRAb first international standardpreparation (90/672) was obtained from the National Institute forBiological Standards and Control (NIBSC; Potters Bar, EN6 3QH, UK).

Inhibition of ¹²⁵I-TSH and ¹²⁵I-mouse TSHR MAb binding to the TSHreceptor

Binding inhibition assays were carried out using TSH receptor coatedtubes as described previously (J Sanders, Y Oda, S Roberts, A Kiddie, TRichards, J Bolton, V McGrath, S Walters, D Jaskolski, J Furmaniak, BRees Smith; “The interaction of TSH receptor autoantibodies with¹²⁵I-labeled TSH receptor”; Journal of Clinical Endocrinology andMetabolism 1999; 84: 3797-3802) (reagents from RSR Ltd). Briefly, 100 μLof sample (tissue culture supernatant, purified IgG or Fab fragment,patient serum or NIBSC 90/672 standards) were incubated in TSH receptorcoated tubes at room temperature for 2 hours with gentle shaking. Afteraspiration, the tubes were washed twice with 1 mL of assay buffer (50mmol/L NaCl, 10 mmol/L Tris-HCl pH 7.8, 0.1% Triton X-100) beforeaddition of 100 μL of ¹²⁵I-TSH or ¹²⁵I-MAb (5×10⁴ cpm) and incubation atroom temperature for 1 hour with shaking. The tubes were then washedtwice with 1 mL of assay buffer, aspirated and counted in a gammacounter.

Inhibition of binding was calculated as:100×1−cpm bound in the presence of text material/cpm bound in thepresence of control material

Control materials used were culture medium, a pool of healthy blooddonor sera or as otherwise indicated.

Analysis of Thyroid Stimulating Activities

The ability of monoclonal autoantibody preparations and patient sera tostimulate the production of cyclic AMP (or cAMP) in CHO cells expressinghTSH receptor (approximately 50,000. receptors per cell) (Y Oda, JSanders, S Roberts, M Maruyama, R Kato, M Perez, V B Petersen, NWedlock, J Furmaniak, B Rees Smith; “Binding characteristics ofantibodies to the TSH receptor”; Journal of Molecular Endocrinology1998; 20: 233-244) were carried out according to the method of R Latif,P Graves, T F Davies; “Oligomerization of the human thyrotropinreceptor”; Journal of Biological Chemistry 2001; 276: 45217-45224.Briefly, CHO cells were seeded into 96 well plates (30,000 cells perwell) and incubated for 24 hours in DMEM (Invitrogen Ltd; Paisley PA49RF, UK) containing 10% fetal calf serum. Culture was then continued inDMEM without fetal calf serum for a further 24 hours. The DMEM was thenremoved and test TSH, IgG, Fab and serum (100 μl diluted in NaCl freeHank's Buffered Salts solution containing Ig/L glucose, 20 mmol/L Hepes,222 mmol/L sucrose, 15 g/L bovine serum albumin (BSA) and 0.5 mmol/L 3isobutyl-1-methyl xanthine pH 7.4) added and incubated for 1 hour at 37°C. After removal of the test solutions, cells were lysed and assayed forcyclic AMP using a Biotrak enzyme immunoassay system from AmershamBiosciences; Chalfont St Giles, HPS 4SP, UK. In some experiments, theability of patient sera and mouse monoclonal antibodies to the TSHR toinhibit the stimulating activity of TSH or hMAb TSHR1 was assessed. Thiswas carried out by comparing (a) the stimulatory effects of TSH or hMAbTSHR1 alone with (b) the stimulatory effects of TSH or hMAb TSHR1 in thepresence of patient sera or mouse monoclonal antibody.

Variable Region Gene Analysis

Total RNA was prepared from 1×10⁷ cells of a TSH receptor autoantibodyproducing clone using the acid phenol guanidine method (P Chomczynski, NSacchi; “Single-step method of RNA isolation by acid guanidiniumthiocyanate-phenol-chloroform extraction”; Analytical Biochemistry 1987;162: 156-159) and mRNA prepared using oligo dT magnetic beads (DynalBiotech Ltd; Win-al, CH62 3QL, UK). RT-PCR reactions were performedusing reagents from Invitrogen Ltd; Paisley PA4 9RF, UK.

Sense strand oligonucleotide primers were designed using the sequencesrecommended by the Medical Research Council's V-base database(www.mrc-cpe.cam.ac.uk). Antisense primers specific for human IgG1 heavychain and lambda light chain were based on constant region encoding DNAsequences. Both sense and antisense primers included additional 5′restriction endonuclease site sequences to facilitate cloning of PCRproducts. IgG1 heavy chain and lambda light chain RT-PCR reactions wereperformed using the complete panel of appropriate primers. All primerswere synthesized by Invitrogen Ltd. The RT reaction took place at 50° C.for 10 minutes followed immediately by 40 cycles of PCR (15 sec 94° C.,30 sec 55° C., 30 sec 72° C.). RT-PCR products were cloned into pUC18and DNA prepared using the Wizard kit from Promega UK Ltd; SouthamptonSO 16 7NS, UK and sequenced by the Sanger-Coulson method (F Sanger, SNicklen, A R Coulson; “DNA sequencing with chain terminatinginhibitors”; Proceedings of the National Academy of Sciences of the USA1977; 74: 5463-5467). V region sequences were compared with availablesequences of human Ig genes using Ig blast(www.ncbi.nlm.nih.gov/igblast/igblast.cgi).

Immunoprecipitation Assay (IPA)

The cDNA encoding full length TSH receptor was placed downstream of theT7 promoter in pYES2 (Invitrogen) and used in an in vitro TnT system(Promega UK Ltd) to produce TSH receptor labelled with ³⁵S-methionine aspreviously described (L Prentice, J Sanders, M Perez, R Kato, J Sawicka,Y Oda, D Jaskolski, J Furmaniak, B Rees Smith; “Thyrotropin (TSH)receptor autoantibodies do not appear to bind to the TSH receptorproduced in an in vitro transcription/translation system”; Journal ofClinical Endocrinology and Metabolism 1997; 82: 1288-1292). Briefly 50μL ³⁵S-labelled TSH receptor (25 000-30 000 cpm) diluted in HSB (150mmol/L Tris-HCL pH 8. 3.200 mmol/L NaCl and 10 mg/mL bovine serumalbumin containing 1% Tween 20) were added to duplicate 50 μL aliquotsof diluted test sample and incubated for 2 hours at room temperature.Immune complexes were then precipitated by addition of protein Asepharose (Sigma-Aldrich) and counted in a scintillation counter.

TSH Receptor Preparations and Western Blotting

Full-length human TSH receptor was expressed in CHO-K1 cells, extractedwith 1% Triton X-100 and purified by TSH receptor monoclonal antibodyaffinity chromatography as described previously (Y Oda, J Sanders, MEvans, A Kiddie, A Munkley, C James, T Richards, J Wills, J Furmaniak, BRees Smith; “Epitope analysis of the human thyrotropin (TSH) receptorusing monoclonal antibodies”; Thyroid 2000; 10:1051-1059).

The purified CHO cell produced TSH receptor was run on 9% SDS-PAGE gels,blotted onto nitrocellulose and reacted with test antibodies asdescribed previously (Y Oda, J Sanders, M Evans, A Kiddie, A Munkley, CJames, T Richards, J Wills, J Furmaniak, B Rees Smith; “Epitope analysisof the human thyrotropin (TSH) receptor using monoclonal antibodies”;Thyroid 2000; 10: 1051-1059).

Epitope Analysis Using TSH Receptor Peptides

Twenty six peptides each 25aa long covering the whole of theextracellular domain of the human TSH receptor were kindly provided byDr J Morris (J C Morris, E R Bergert, D J McCormick; “Structure-functionstudies of the human thyrotropin receptor. Inhibition of binding oflabeled thyrotropin (TSH) by synthetic human TSH receptor peptides”;Journal of Biological Chemistry 1993; 268: 10900-10905). A human 21-OHpeptide (C1, SSSRVPYKDRARLPL) which binds to an M21-OH5 MAb (S Chen, JSawicka, L Prentice, J F Sanders, H Tanaka, V Petersen, C Betterle, MVolpato, S Roberts, M Powell, B Rees Smith, J Furmaniak; “Analysis ofautoantibody epitopes on steroid 21-hydroxylase using a panel ofmonoclonal antibodies”; Journal of Clinical Endocrinology and Metabolism1998; 83: 2977-2986) was used as a positive control and a humanmonoclonal antibody to GAD₆₅ (N Hayakawa, L D K E Premawardhana, MPowell, M Masuda, C Arnold, J Sanders, M Evans, S Chen, J C Jaume, SBaekkeskov, B Rees Smith, J Furmaniak; “Isolation and characterizationof human monoclonal autoantibodies to glutamic acid decarboxylase”;Autoimmunity 2002; 35: 343-355) was used as a negative control. Thepeptide ELISA was carried out as described previously (Y Oda, J Sanders,M Evans, A Kiddie, A Munkley, C James, T Richards, J Wills, J Furmaniak,B Rees Smith; “Epitope analysis of the human thyrotropin (TSH) receptorusing monoclonal antibodies”; Thyroid 2000; 10: 1051-1059).

Interaction of Monoclonal TSHR Autoantibody Preparations with the TSHReceptor Coated onto Plastic Tubes or ELISA Plate Wells

(a) ¹²⁵I-Labelled Autoantibody

Test samples including patient sera (100 μL) were incubated in TSHreceptor coated tubes (RSR Ltd.) at room temperature for 2 hours withgentle shaking. After aspiration, the tubes were washed twice with 1 mLof assay buffer before addition of 100 μL of labelled autoantibodypreparation (30,000 cpm) and incubation at room temperature for 1 hourwith shaking. The tubes were then washed twice with 1 mL of assaybuffer, aspirated and counted in a gamma counter. Inhibition of¹²⁵I-labelled autoantibody binding was calculated using the formula asfor inhibition of TSH binding (see above).

(b) Biotin Labelled Monoclonal Autoantibody and Biotin Labelled TSH

The procedure described previously (J Bolton, J Sanders, Y Oda, CChapman, R Konno, J Furmaniak and B Rees Smith; “Measurement ofthyroid-stimulating hormone receptor autoantibodies by ELISA; ClinicalChemistry, 1999; 45: 2285-2287) was used. Briefly, test samplesincluding patient sera (75 μL) were incubated in TSH receptor coatedELISA plate wells (RSR Ltd) for 2 hours with shaking (200 shakes perminute) on an ELISA plate shaker. Test samples were then removed and thewells washed once with assay buffer. Biotin-labelled monoclonal TSHreceptor autoantibody (1 ng in 100 μL) or biotin labelled porcineTSH(RSR Ltd; 5 ng in 100 μL) were then added and incubation continuedfor 25 minutes at room temperature without shaking. The wells werewashed once, 100 μL of streptavidin-peroxidase (RSR Ltd; 10 ng in 100μL) added and incubation continued for 20 minutes at room temperaturewithout shaking. The wells were then washed 3 times, the peroxidasesubstrate tetramethyl benzidine (RSR Ltd, 100 μL) added. Afterincubation for 30 minutes at room temperature without shaking 50 μL of0.5M H₂SO₄ was added to stop the substrate reaction and the absorbanceof each well read at 450 nm on an ELISA plate reader. Inhibition ofbiotinylated MAb or TSH binding was expressed as an index calculated as:100×1−text sample absorbance at 450nm/negative serum control absorbanceat 450nmScatchard Analysis of Monoclonal Autoantibody Binding to TSH ReceptorCoated Tubes

Unlabelled IgG or Fab in 50 μL of assay buffer and 50 μL of¹²⁵I-labelled hMAb IgG or Fab (30,000 cpm in assay buffer) wereincubated for 2 hours at room temperature with gentle shaking, washedtwice with 1 mL of assay buffer and counted in a gamma counter. Theconcentration of IgG or Fab bound vs bound/free was plotted (GScatchard; “The attraction of proteins for small molecules and ions”;Annals of the New York Academy of Sciences 1949; 51: 660-672) to derivethe association constants.

Binding of TSH Receptor to Tubes Coated with Monoclonal TSH ReceptorAutoantibodies

Test samples including patient sera (100 μl) and detergent solubilisedTSH receptor (20 μL) were incubated for 1 hour at room temperature.Duplicate 50 μL aliquots of the incubation mixture were then added toplastic tubes (Nunc Maxisorp) which had been coated with monoclonal TSHreceptor autoantibody Fab (200 μL of 10 μg/mL overnight at 4° C.followed by washing and post coating). After incubation for 1 hour atroom temperature with gentle shaking, the tubes were washed, 100 μL(40,000 cpm) of ¹²⁵I-labelled TSH receptor C terminal monoclonalantibody 4E31 (J Sanders, Y Oda, A Kiddie, T Richards, J Bolton, VMcGrath, S Walters, D Jaskolski, J Furmaniak, B Rees Smith; “Theinteraction of TSH receptor autoantibodies with ¹²⁵I-labelled TSHreceptor”; Journal of Clinical Endocrinology and Metabolism 1999; 84:3797-3802) added and incubation continued for a further 1 hour withgentle shaking. Then tubes were washed and counted for ¹²⁵I.

Cloning and Expression of Recombinant hMab TSHR1 Fab in E coli

The hMAb TSHR1 heavy chain RT-PCR product (see Variable Region GeneAnalysis section) was cut with XhoI and Spe1 restriction endonucleasesand the hMAb TSHR1 light chain PCR product was cut with Sad and XbaIrestriction endonucleases and both heavy and light chain cDNAs clonedinto the Immunozap H/L vector (Stratagene Europe; Amsterdam,Netherlands) (I Matthew, G Sims, S Ledwidge, D Stott, D Beeson, NWillcox, A Vincent; “Antibodies to acetylcholine receptor in parouswomen with myasthenia: evidence for immunization by fetal antigen”;Laboratory Investigation 2002; S2: 1-11) under the control of the lacZpromoter. Plasmid DNA was prepared using the Qiagen midi plasmidpurification kit (Qiagen Ltd; Crawley, RH10 9AX, UK) and the presence ofhMAb TSHR1 heavy and light chain cDNAs confirmed by sequencing using theSanger-Coulson method (F Sanger, S Nicklen, A R Coulsen; “DNA sequencingwith chain terminating inhibitors”; Proceedings of the National Academyof Sciences of the USA 1977; 74: 5463-5467). Plasmid DNA was transformedinto 2 different E coli strains (a) XL1-Blue MRF′ (Stratagene) and (b)HB2151 (Amersham Biosciences) and grown overnight at 37° C. on LBampicillin (Tryptone 10 g/L, Yeast Extract 5 g/L, NaCl 10 g/L, 100 μg/mLfinal concentration Ampicillin) agar plates (15 g/L agar). Precultures(one colony in 3 mL LB ampicillin+1% glucose) were grown overnight at37° C. with shaking. Production of the recombinant Fab is inhibited inthe presence of glucose. Precultures after overnight incubation werediluted 1/100 (0.5 mL in 50 mL LB ampicillin) and grown at 37° C. untilthe OD₆₀₀ was between 0.4-0.6. These cultures were placed at 30° C. withshaking for 20 minutes. Thereafter isopropyl-β-D thiogalactoside (IPTG)was added to a final concentration of 1 mmol/L and cultures continued tobe incubated overnight (16 hours) at 30° C. with shaking. The cultureswere then centrifuged at 3000 rpm for 30 minutes at 4° C. and theculture supernatants and pellets recovered. The pellets, wereresuspended in 1 mL of ice cold TES buffer (0.2 mol/L Tris-HCl pH 8.0,0.5 mol/L EDTA and 0.5 mol/L sucrose) by vortexing. A further 1.5 mL ofice cold TES buffer diluted 5× in H₂ 0 was added, the mixture vortexedagain and incubated on ice for 30 minutes then recentrifuged to give asecond supernatant or periplasmic fraction (PF). The culture supernatantand PF were filtered through a 0.45 μm filter and dialysed overnightinto 10 mmol/L Tris pH 7.5, 50 mmol/L NaCl. Culture supernatant or PFfrom untransformed XL1-Blue MRF′ and HB2151 cells and XL1-Blue MRF′transformed with hMAb TSHR1 plasmid (XL1-Blue MRF′/hMAb TSHR1) andHB2151 transformed with hMAb TSHR1 plasmid (HB2151/hMAb TSHR1) grownwith glucose without IPTG ie non-induced were also prepared. The culturesupernatants and PF were assayed for (a) their ability to inhibit TSHbinding to the TSHR, (b) their ability to stimulate cyclic AMPproduction in CHO cells expressing TSHR, and (c) total recombinant Fabconcentration by radioimmunoassay. In this assay, calibrators and testmaterials including culture supernatants and PF (100 μL in duplicate)diluted in assay buffer (50 mmol/L NaCl, 10 mmol/L Tris-HCl pH 7.8, 0.1%Triton X-100) were incubated for 1 hour at room temperature in plastictubes coated with Fab specific goat anti human IgG (from Sigma Aldrich,Poole, BH12 4QH, UK). The tubes were then washed with assay buffer (2×1mL) and 1004, of hMAb TSHR1 Fab (30,000 cpm) added followed byincubation at room temperature. After 1 hour, the tubes were washedagain (2×1 mL) and counted for ¹²⁵I. The counts bound were plottedagainst concentration of Fab (hybridoma produced hMAb TSHR1 Fab) in thecalibrators (5-500 ng/mL) and the concentration of recombinant Fab inthe various test materials read off this calibration curve. Thedetection limit for this assay was 5-10 ng/mL of Fab.

Cloning and Expression of Recombinant 4B4 (a Human MAb to Glutamic AcidDecarboxylase or GAD) and Recombinant Hybrid Fabs (Mixed HC and LC ofhMAb TSHR1 and 4B4)

Recombinant 4B4 Fab (4B4 is described in detail by N Hayakawa, L D K EPremawardhana, M Powell, M Masuda, C Arnold, J Sanders, M Evans, S Chen,J C Jaume, S Baekkeskov, B Rees Smith, J. Furmaniak. Isolation andcharacterization of human monoclonal antibodies to glutamic aciddecarboxylase. Autoimmunity 2002 volume 35 pp 343-355) and recombinanthybrid Fabs were produced by cloning the respective HC and LC intoImmunozap H/L vector and expressed in HB2151 cells as described forrecombinant hMAb TSHR1 Fab. The culture supernatants and periplasmicfraction were assayed for their ability to (a) inhibit TSH binding tothe TSHR (b) to stimulate cyclic AMP production in CHO cells expressingthe TSHR and (c) for total recombinant Fab concentration as describedabove. In addition, GAD Ab activity was assessed as described below.

Measurement of Recombinant GAD Ab Fab Activity in Culture Supernatantsand Periplasmic Fractions

An assay based on the ability of GAD Ab Fab preparation to inhibit thebinding of ¹²⁵I-labelled GAD (from RSR Ltd, Cardiff, CF23 SHE, UK) tothe human monoclonal antibody to GAD (4B4) was used. In the assay, testsamples diluted in GAD Ab assay buffer (150 mmol/L NaCl, 50 mmol/LTris-HCl pH 8.0, 1% v/v Tween 20, 1 g/L bovine serum albumin and 0.5 g/LNaN₃) were incubated (50 μL in duplicate with ¹²⁵I-labelled GAD (30,000cpm in 50 μL of GAD Ab assay buffer) for 1 hour at room temperature.Then, 50 μL of 4B4 IgG (0.1 μg/mL in GAD Ab assay buffer) was added andincubation continued for 24 hours at room temperature. Solid phaseprotein A (50 μL in GAD Ab assay buffer; from RSR Ltd) was then added toprecipitate complexes of 4B4 IgG-¹²⁵I-labelled GAD (protein A does notreact with complexes of Fab and ¹²⁵I-labelled GAD). After allowing thereaction with protein A to proceed for 1 hour at room temperature, theprecipitates were pelleted by centrifugation (1500 g for 30 minutes at4° C.), washed with 1 mL of GAD Ab assay buffer and counted for ¹²⁵I.¹²⁵I-labelled GAD binding in the absence of 4B4 IgG was 4-5% of thetotal cpm added.

Production of Anti-Idiotypic Antibodies to hMAb TSHR1

6-8 week old BALB/c mice were immunised intraperitoneally with 50 μghMAb TSHR1 Fab in complete Freunds adjuvant followed by a secondinjection with 50 μg hMAb TSHR1 Fab in incomplete Freunds adjuvant after25 days and a further injection of 50 μg hMAb TSHR1 Fab 4 days beforeremoval of the spleen. The spleen cells from antibody positive mice (seebelow) were fused with a mouse myeloma cell line and monoclonalantibodies isolated as above for TSHR MAbs. The levels of anti-idiotypicantibody in the mouse sera and cell culture wells were measured byinhibition of ¹²⁵I-hMAb TSHR1 Fab binding to TSHR coated tubes. Inparticular, duplicate 60 μL aliquots of test sample (diluted in assaybuffer: 50 mmol/L NaCl, 10 mmol/L Tris-HCl pH 7.8, 0.1% Triton X-100)were incubated with 60 μL of ¹²⁵I-hMAb TSHR1 Fab (30 000 cpm diluted inassay buffer) for 1 hour at room temperature. 100 μL of the mixture wastransferred to duplicate TSHR coated tubes (RSR Ltd) with 20 μL startbuffer (see above) and incubation continued for a further two hours atroom temperature with shaking. The tubes were then washed with 2×1 mL ofassay buffer and counted for ¹²⁵I. The presence of anti-idiotypicantibodies reactive with hMAb TSHR1 was evident from the ability of testsamples to inhibit the binding of labelled hMAb TSHR1 Fab to the TSHRcoated tubes.

Results

Lymphocytes (30×10⁶) obtained from 20 mL of patient's blood were platedout at 1×10⁶ per well on a 48 well plate with 200 μL of EBV supernatanton feeder layers of mouse macrophages. On day 11 post EBV infection thesupernatants were monitored for inhibition of ¹²⁵I-TSH binding. One wellwas found to be positive for inhibition of binding, the levels ofinhibition increasing to greater than 90% inhibition by day 16 andstayed at that level until day 24, after which time they decreased. Thecultures were expanded and fused with K6H6/B5 cells on day 21,23, 26 and27 post EBV infection; in total 7 fusion experiments were carried out.Each fusion was plated across 3×96 well plates (ie 21 plates in total)and one well, stably producing antibodies with ¹²⁵I-TSH bindinginhibiting activity was obtained. This was followed by 3 rounds ofre-cloning to yield a single clone producing a human monoclonal antibodywhich inhibited labelled TSH binding to the TSH receptor. This humanmonoclonal TSH receptor autoantibody was designated hMAb TSHR1 and wasof subclass IgG1 with a lambda light chain.

The ability of different concentrations of hMAb TSHR1 IgG and Fab toinhibit labelled TSH binding to the TSH receptor is shown in FIG. 1. Ascan be seen in FIG. 1 as little as 1 ng/mL of these preparationsinhibited TSH binding with more than 90% inhibition being obtained with1000 ng/mL. TSMAb TSHR1 IgG and Fab also stimulated cyclic AMPproduction in CHO cells transfected with the TSH receptor as shown inFIG. 2. As little as 1 ng/mL of hMAb TSHR1 IgG or Fab caused strongstimulation of cyclic AMP. Similar levels of stimulation were observedwith 0.1 ng/mL porcine TSH and 10 ng/mL of human TSH. Comparison of theability of the serum from the original lymphocyte donor (taken at thesame time as the blood sample for lymphocyte isolation) to inhibitlabelled TSH binding to the TSH receptor and to stimulate cyclic AMPproduction in TSH receptor transfected CHO cells is shown in FIG. 3A.Inhibition of TSH binding could be detected with serum diluted 500×whereas stimulation of cyclic AMP could be detected with serum diluted5000×.

¹²⁵I-labelled hMAb TSHR1 IgG bound to TSH receptor coated tubes andScatchard analysis indicated an association constant of 5×10¹⁰ molar⁻¹.This binding was inhibited by sera from patients with Graves' diseasewho had TSH receptor autoantibodies (detectable by inhibition oflabelled TSH binding) (Table 1), ¹²⁵I-labelled hMAb TSHR1 Fab also boundto TSH receptor coated tubes (association constant by Scatchardanalysis=4.5×10¹⁰ molar⁻¹) and this binding was inhibited by TSHreceptor autoantibody positive Graves' sera (Table 2). In addition,detergent solubilised preparations were able to bind to plastic tubescoated with hMAb TSHR1 and this binding could be inhibited by seracontaining TSH receptor autoantibodies (Table 3).

As shown in Table 4 hMAb TSHR1-biotin bound to TSH receptor coated ELISAplates and the binding was inhibited by the international referencepreparation NIBSC 90/672 and serum from patients with Graves' disease.Inhibition of binding was not observed by sera from healthy blooddonors. FIG. 3B shows a graphical representation of a comparison betweenan assay for TSHR autoantibodies based on hMAb TSHR1-biotin and earlierassays. The sensitivity of the assay based on hMAb TSHR1-biotin isclearly superior according to concentration of the internationalstandard NIBSC 90/672 detectable. This was confirmed in a study of serafrom 72 patients with Graves' disease shown in FIG. 3C. Healthy blooddonor sera (n=100) and sera from subjects with non-thyroid diseases(n=43) gave respectively values of up to 10% inhibition of hMAb TSHR1binding and up to 11% inhibition of TSH binding in this study.

hMAb TSHR1 IgG did not react with full length TSH receptor preparationson Western blot analysis nor did it react well with ³⁵S-labelled fulllength TSH receptor in the immunoprecipitation assay nor in the TSHreceptor peptide ELISA. This lack of reactivity indicates that hMAbTSHR1 reacts with conformational rather than linear epitopes on the TSHreceptor.

Sequence analysis of the genes coding for hMAb TSHR1 indicated that theheavy chain V region genes were of the VH5 family, the D gene of theD6-13 family and the J gene of the JH5 family and for the light chainthe V-gene region is from the VL1-11 germline and the J-gene region isfrom the JL3b germline. The heavy chain nucleotide and amino acidsequences are shown in FIGS. 4 and 5 respectively and the light chainnucleotide and amino acid sequences are shown in FIGS. 6 and 7respectively. These sequences are a refinement of the HC and LCnucleotide sequences determined using PCR primers which are degenerate.In particular a HC sequencing artefact for nucleotides 115-120 wasidentified. Sequencing indicated cacgtg (transcribed to amino acids HisVal) whereas the crystal structure more reliably indicated amino acidsGln Leu (corresponding bases being cagctg). Crystal structure analysisalso enabled refinement of the HC and LC derived amino acid sequencesparticularly in the degenerate PCR primer region. In the case of the LCamino acid 2 was found to be Pro by RT-PCR but was Thr from the crystalstructure. In the case of the HC amino acid 2 was found to be Met byRT-PCR but was Val from the crystal structure.

Comparison of the activities of hMAb TSHR1 IgG preparations and theinternational standard for TSH receptor autoantibodies in terms ofinhibition of labelled TSH binding are shown in Table 5. This enabled aspecific activity of hMAb TSHR1 IgG to be estimated as 138 units ofNIBSC 90/672 per mg of protein when the assays were carried out in serumand 163 units per mg when the assays were carried out in assay buffer(mean of the 2 values=150 units/mg). hMAb TSHR I Fab preparations were288 and 309 units per mg in serum and assay buffer respectively (mean ofthe 2 values=300 units/mg). Table 6 shows a similar analysis of thelymphocyte donor serum and the donor serum IgG. As can be seen the donorserum contains a mean of 0.38 units/mL of NIBSC 90/672 (0.36 and 0.4 inserum and assay buffer respectively) and the donor serum IgG has a meanspecific activity of 0.059 units per mg of protein. These results aresummarised in Table 7 and comparison with the specific activity of hMAbTSHR1 IgG (150 units/mg) indicates that the monoclonal autoantibody IgGis 2500 times more active than the lymphocyte donor serum IgG in termsof inhibition of TSH binding.

Initial assessment of the activities of the various IgG and serumpreparations in terms of stimulation of cyclic AMP in CHO cellstransfected with the TSH receptor are also shown in Table 7. Thestimulation of cyclic AMP assay is characterized by considerable withinassay and between assay variability. This relates to several factorsincluding variation in the number and quality of cells initially seededinto the 96 well plates and variation in growth rate of the seeded cellsover the subsequent 48 hours. Consequently the assays of hMAb TSHR1 IgGand Fab, lymphocyte donor serum and serum IgG and NIBSC 90/672 werecarried out repeatedly and the results are summarized in Table 8. Thespecific activity of the hMAb TSHR1 IgG was 318 units per mg in thestimulation assay compared with 0.1 units per mg for the lymphocytedonor serum IgG ie the monoclonal autoantibody IgG was about 3000 timesas active as the donor serum IgG in terms of stimulation of cyclic AMPproduction. This value is in reasonable agreement with the value of 2500times observed for inhibition of TSH binding measurements (see above andTables 5 and 6). Table 9 shows a further analysis of the TSH receptorstimulating effects of hMAb TSHR1 IgG and Fab and the lymphocyte donorserum IgG.

The effects of porcine TSH and hMAb TSHR1 IgG on stimulation of cyclicAMP production in CHO cells expressing the TSH receptor were additive ascan be seen in the results shown in Table 10.

Typical results observed in the stimulation of cyclic AMP assay with thereference preparation NIBSC 90/672 are shown in Table 11.

Tables 12 and 13 show the effects of the various E. coli culturesupernatants in terms of inhibition of labelled TSH binding andstimulation of cyclic AMP production respectively. Transformed (withhMAb THSR1 plasmid) and IPTG induced cultures of both strains of E. coliproduced sufficient amounts of recombinant hMAb TSHR Fab to act aspotent inhibitors of TSH binding (Table 12) and powerful stimulators ofcyclic AMP production (Table 13). Control culture supernatants (fromuntransformed cells and transformed but non-induced cells) did notproduce detectable levels of binding inhibition (Table 12) orstimulating (Table 13) activities.

In further control experiments, a recombinant human antibody Fabproduced by cloning and expression of the HC and LC of a humanmonoclonal antibody to GAD (4B4) were analysed. Assays of culturesupernatants and periplasmic fractions indicated that recombinant 4B4Fab did not have detectable inhibition of TSH binding activity (Tables14 and 15) or TSHR stimulating activity (Tables 16 and 17). Furthermore,hybrid Fabs consisting of (a) hMAb TSHR1 HC and 4B4 LC (b) hMAb TSHR1 LCand 4B4 HC did not show interaction with the TSHR in either assay(Tables 14-17). Assays for GAD Ab activity in these various recombinantFab preparations were only able to detect GAD Ab expression in cellstransformed with 4B4 HC and 4B4 LC (Tables 18 and 19). Recombinant hMAbTSHR1 Fab did not show detectable GAD Ab activity and neither didrecombinant Fab hybrids consisting of mixtures of 4B4 and hMAb TSHR1 HCand LC (Tables 18 and 19).

The ability of hMAb TSHR1 to stimulate cyclic AMP production in CHOcells transfected with the TSHR was inhibited by patient sera containingTSHR autoantibodies which acted as TSH antagonists (FIG. 8). Inaddition, a mouse monoclonal antibody to the TSHR (9D33) was able toblock hMAb TSHR1 stimulating activities (Table 20) whereas another TSHRmouse MAb (2B4) was ineffective (Table 20). However 2B4 was able toblock the stimulating activities of TSH as was 9D33 (Table 21). Table 22shows the ability of 2B4 and 9D33 to inhibit the binding of¹²⁵I-labelled TSH and ¹²⁵I-labelled hMAb TSHR1 to TSHR plastic tubes.9D33 was able to inhibit labelled TSH binding and labelled hMAb TSHR1binding quite effectively (more than 50% inhibition at 10 μg/mL). 2B4was an effective inhibitor of labelled TSH binding to the TSHR (morethan 80% inhibition at 1 μg/mL) but had only a minor effect on hMAbTSHR1 binding (11% inhibition at 1 μg/mL) or on 9D33 binding (22%inhibition at 1 μg/mL). The binding of labelled 9D33 to TSHR coatedtubes was inhibited by sera from patients with Graves' diseasecontaining TSHR autoantibodies (as measured by inhibition of labelledTSH binding to the TSHR) whereas healthy blood donor sera and sera frompatients with other autoimmune diseases had little or no effect (Table23). Labelled 9D33 binding to the TSHR was inhibited by TSHRautoantibodies with TSH agonist or antagonist properties (Table 24) andby the international standard NIBSC 90/672 (Table 25). Scatchardanalysis indicated that 9D33 and 2B4 had affinities of 2×10¹⁰ molar⁻¹and 1×10¹⁰ molar⁻¹ respectively for TSH receptors coated onto plastictubes.

Immunisation of mice with hMAb TSHR1 Fab resulted in production ofantibodies in the mice sera (polyclonal antibodies) which were capableof binding to hMAb TSHR1 Fab in such a way as to inhibit the ability ofthe Fab to bind to the TSHR (Table 26). Furthermore, a monoclonalantibody produced from the spleen cells of a mouse immunised with hMAbTSHR1 Fab was also able to inhibit the Fab binding to the TSHR (Table27).

Overall our analysis indicates that the human monoclonal autoantibodyhMAb TSHR I has the TSH receptor binding and thyroid stimulatingcharacteristics of the TSH receptor autoantibodies in the serum of thelymphocyte donor. As detailed above, the monoclonal antibody was alsoproduced as a recombinant Fab preparation.

CONCLUSIONS

-   (a) We have produced a human monoclonal autoantibody to the TSH    receptor which has similar properties to the TSH receptor    autoantibody in the donor patient's serum. The monoclonal antibody    was also produced as a recombinant Fab preparation.-   (b) The monoclonal antibody IgG and Fab and recombinant Fab    preparations are powerful thyroid stimulators and effective    inhibitors of labelled TSH binding to the TSH receptor.-   (c) Binding of labelled MAb IgG and Fab preparations to the TSH    receptor is inhibited by TSH receptor autoantibody positive sera    from patients with Graves' disease but not by healthy blood donor    sera or sera from patients with other autoimmune diseases. Assay    systems for TSHR autoantibodies based on inhibition of labelled hMAb    TSHR1 binding to the TSH receptor are more sensitive than other    assays so far described.-   (d) TSH receptor autoantibodies which act as TSH antagonists as well    as TSH receptor autoantibodies which act as TSH agonists inhibit    labelled hMAb TSHR1 binding to the TSH receptor.-   (e) hMAb TSHR1 preparations coated onto plastic tubes bind TSH    receptor and this binding is inhibited by TSH receptor    autoantibodies in different patient sera.-   (f) A mouse monoclonal antibody (9D33) which inhibits hMAb TSHR1    binding to the TSHR was also found to block the stimulating activity    of hMAb TSHR1 and TSH.-   (g) Mouse polyclonal and monclonal antibodies to hMAb TSHR1 have    been produced which bind to hMAb TSHR1 in such a way as to prevent    it binding to the TSH receptor. These anti-idiotypic antibodies    compete therefore with the TSHR for hMAb TSHR1 and as such they may    be useful alternatives to the TSHR in applications where a binding    partner for TSHR autoantibodies is required.-   (h) These results indicate that hMAb TSHR1 and/or its derivatives    and/or its competitors can be used as a replacement for TSH in-   (i) assays for TSH receptor autoantibodies, TSH and related ligands-   (ii) various in vivo applications involving provision of TSH agonist    or TSH antagonist activities.-   (iii) identification and provision of new types of TSH receptor    autoantibody binding sites.

TABLE 1 Effect of patient sera on ¹²⁵I-labelled hMAb TSHR1 IgG bindingto the TSH receptor and comparison with effect on ¹²⁵I-labelled TSHbinding to the TSH receptor inhibition inhibition of labelled oflabelled hMAb inhibition hMAb inhibition Test TSHR1 of TSH Test TSHR1 ofTSH material binding binding material binding binding G1 62 80 N1 3.17.7 G2 91 93 N2 2.4 2.6 G3 91 76 N3 −1.0 4.5 G4 94 92 N4 −11 6.5 G5 9394 N5 1.7 5.0 G6 76 85 N6 2.8 1.7 G7 87 90 N7 5.2 −0.8 G8 65 45 N8 3.50.2 G9 88 90 N9 2.8 −0.6 G10/10 83 59 N10 4.5 2.2 G10/20 69 43 D1 −4.82.2 G10/40 56 29 A1 −3.1 1.3 G10/80 42 19 A2 −3.5 −3.0 G11/10 75 73G11/20 59 54 G11/40 39 33 G11/80 22 18 G1-G11 are sera from patientswith a history of Graves' disease. G9 serum has high levels of TSHblocking (ie TSH antagonist activity). G10 and G11 have high levels ofthyroid stimulating activity. G10 is the lymphocyte donor serum. /10,/20 etc indicate dilution factor in a pool of healthy blood donor sera.N1-N10 are sera from healthy blood donors. D1 is from a patient withtype 1 diabetes mellitus (positive for autoantibodies to glutamic aciddecarboxylase). A1 and A2 are from patients with Addison's disease(positive for steroid 21-hydroxylase autoantibodies). In the presence ofthe pool of healthy blood donor sera about 25% of ¹²⁵I-labelled MAb IgGbound to the TSHR coated tubes.

TABLE 2 Effect of patient sera on ¹²⁵I-labelled hMAb TSHR1 Fab bindingto the TSH receptor and comparison with effect on ¹²⁵I-labelled TSHbinding to the TSH receptor inhibition of labelled Fab inhibition ofTest material binding TSH binding NIBSC 90/672 diluted in a pool ofhealthy blood donor serum to 1 U/L 17 13 to 2 U/L 27 24 to 4 U/L 47 44to 8 U/L 61 65 Healthy blood donor serum A −3 <10 Healthy blood donorserum B 3 <10 Healthy blood donor serum C 4 <10 Healthy blood donorserum D −4 <10 Healthy blood donor serum E 0 <10 Graves' serum F 64 78Graves' serum G 42 54 Graves' serum H 49 69 Graves' serum I 24 36Graves' serum J 76 88

TABLE 3 Binding of TSHR to plastic tubes coated with hMAb TSHR 1 Fab andinhibition of TSHR binding by sera containing TSHR autoantibodies Testmaterial cpm bound¹ Healthy blood donor serum A 8406 Healthy blood donorserum B 8430 TSHR autoantibody positive serum 1 1527 TSHR autoantibodypositive serum 2 1131 TSHR autoantibody positive serum 3 1199 ¹TSHRbinding was detected using a ¹²⁵I-labelled mouse monoclonal antibody tothe TSHR C terminus; total cpm = 39,000 per tube.

TABLE 4 Effect of patient serum samples on binding of biotin labelledhMAb TSHR1 and biotin labelled TSH to ELISA plate wells coated with TSHRhMAb TSHR1 biotin TSR biotin OD₄₅₀ % inhibition OD₄₅₀ % inhibition HBDpool 1.852 0 1.778 0 HBD pool plus 1 U/mL 1.46 21 1.489 16 HBD pool plus2 U/mL 1.168 37 1.304 27 HBD pool plus 4 U/mL 0.792 57 0.947 47 HBD poolplus 8 U/mL 0.539 71 0.492 72 HBD pool plus 40 U/mL 0.118 94 0.233 87Serum P1 1.415 24 1.397 21 Serum P2 1.264 32 1.256 29 Serum P3 0.558 700.408 77 Serum P4 0.763 59 0.907 49 Serum P5 1.047 43 — Serum P6 0.84355 — Serum P7 1.429 23 — HBD 1 1.745 6 1.713 4 HBD 2 1.807 2 — HBD 31.779 4 1.626 9 HBD 4 1.821 2 — HBD 5 1.841 1 1.660 7 HBD 6 1.762 51.777 0 HBD 7 1.799 3 1.767 1 HBD 8 1.783 4 1.703 4 HBD 9 1.792 3 1.6693 HBD = healthy blood donor serum U/mL are units of NIBSC 90/672 SerumP1-P7 are from patients with Graves' disease

TABLE 5 Inhibition of TSH binding by WHO reference preparation NIBSC90/672 and by hMAb TSHR1 IgG and Fab preparations Samples diluted inserum¹ Samples diluted in assay buffer % mean % mean Sample inhibitionunits/L units/mg units/mg inhibition units/L units/mg units/mg NIBSC90/672 0.125 units/L 2 0.25 units/L 4 0.5 units/L 11 1.0 units/L 15 192.0 units/L 28 38 4.0 units/L 48 64 8.0 units/L 69 83 40.0 units/L 95 94hMAb TSHR1 IgG 0 ng/mL 1 0 0.3 ng/mL 1 2 1 ng/mL 3 3 3 ng/mL 7 10 0.4610 ng/mL 21 1.48 148 33 1.73 173 30 ng/mL 46 3.9 130 138 70 4.8 160 163100 ng/mL 81 13.5 135 92 15.6 156 300 ng/mL 92 95 >40 hMAb TSHR1 Fab 0.3ng/mL 5 −2 1 ng/mL 5 1 3 ng/mL 16 1.05 351 16 0.8 265 10 ng/mL 36 2.77277 52 2.9 291 309 30 ng/mL 69 8.0 267 288 86 9.6 372 100 ng/mL 89 23.7237 92 16.9 300 ng/mL 93 94 2G4 IgG² 0.3 ng/mL 2 −3 3 ng/mL 1 −6 30ng/mL 0 −5 300 ng/mL 3 −4 2G4 Fab² 0.3 ng/mL 4 −5 3 ng/mL 4 −6 30 ng/mL1 −5 300 ng/mL 2 −6 ¹Pool of healthy blood donor serum, 14.9% of totalcpm bound to the TSHR in the presence of this serum pool only. 14.7% oftotal cpm bound to the TSHR in the presence of buffer only. ²2G4 is ahuman monoclonal autoantibody to thyroid peroxidase.

TABLE 6 Inhibition of TSH binding by lymphocyte donor serum and donorserum IgG Samples diluted in serum¹ Samples diluted in assay bufferunits/mg units/mg or or (units/mL mean (units/mL mean in units/mg inunits/mg % undiluted or % undiluted or Sample inhibition units/L² serum)(units/mL) inhibition units/L² serum) (units/mL) Donor serum diluted1000× 6 10 diluted 300×  18 1.2 (0.36) 28 1.3 (0.39) diluted 100×  423.2 (0.32) (0.36) 62 3.9 (0.39) (0.40) diluted 30×  78 11.3 (0.39) 9113.5 (0.41) diluted 10×  93 34 95 >40 Donor serum IgG   0 mg/mL 0 0 00.01 mg/mL 7 19 0.87 0.03 mg/mL 23 1.6  0.053 37 1.9  0.063  0.1 mg/mL57 5.1  0.051  0.054 78 6.4  0.064  0.063  0.3 mg/mL 85 17  0.057 93 19 0.063   1 mg/mL 96 43 96 >40 Healthy blood donor pool serum diluted1000× 0 3 diluted 100×  1 4 diluted 10×  1 11 Healthy blood donor poolserum IgG 0.01 mg/mL 2 2  0.1 mg/mL 1 5   1 mg/mL 3 7 ¹Pool of healthyblood donor serum, 14.7% of total cpm bound to the TSHR in the presenceof this serum pool only. 16.3% of total cpm bound to the TSHR in thepresence of buffer only. ²Units shown are NIBSC 90/672 internationalTSHR autoantibody reference preparation.

TABLE 7 Specific activities of hMAb TSHR1 and lymphocyte donor serum andIgG preparations Inhibition of Stimulation of TSH binding assay cyclicAMP assay Units/ Units/ Preparation Units/mg^(1,2) nmole^(1,2) Units/mg¹nmole¹ hMAb TSHR1 IgG 150 22 180 26 hMAb TSHR1 Fab 300 15 700 35 Donorserum IgG 0.059 0.009 0.33 0.048 Donor serum units/mL 0.38 18 ¹Unitsshown are NIBSC 90/672. ²Values are a mean of results obtained in serumand in assay buffer (see Tables 5 and 6)

TABLE 8 Summary of specific activities of hMAb TSHR1 and lymphocytedonor serum and serum IgG determined in several stimulation of cyclicAMP assays Mean Units Number of Standard Preparation per mgdeterminations Deviation hMAb TSHR1 IgG 318 16 189 hMAb TSHR1 Fab 492 10184 Donor serum IgG 0.10 10 0.08 Donor serum units/mL 0.9 4 0.6

TABLE 9 Further analysis of the effects of hMAb TSHR1 IgG and Fab andlymphocyte donor serum IgG in the cyclic AMP stimulation assay Meancyclic number AMP per of Standard Sample well (pmols) determinationsDeviation hMAb TSHR1 IgG 0 ng/mL 0.96 6 0.048 0.3 ng/mL 1.25 6 0.12 1ng/mL 1.84 6 0.16 3 ng/mL 3.4 5 0.37 10 ng/mL 6.6 5 0.62 hMAb TSHR1 Fab0 ng/mL 0.60 6 0.068 0.3 ng/mL 1.11 6 0.11 1 ng/mL 1.99 6 0.39 3 ng/mL4.9 6 0.44 10 ng/mL 10.6 6 0.86 Control human MAb (2G4)¹ IgG 0 ng/mL0.72 11 0.19 IgG 10 ng/mL 0.61 11 0.16 Fab 10 ng/mL 0.61 4 0.044Lymphocyte donor serum IgG 3 μg/mL 1.67 6 0.38 10 μg/mL 4.20 6 0.93 30μg/mL 6.22 6 0.73 Healthy blood donor pool serum IgG 30 μg/mL 0.38 60.10 ¹2G4 is a human monoclonal autoantibody to thyroid peroxidase

TABLE 10 Additive effect of TSH and hMAb TSHR1 IgG in stimulation ofcyclic AMP assays Experiment 1 Experiment 2 cyclic AMP¹ cyclic AMP¹(pmols per (pmols per Sample well) Sample well) A Buffer only 0.57 ABuffer only 0.42 B Porcine TSH 1.07 B Porcine TSH 1.07 0.1 ng/mL 0.05ng/mL C hMAb TSHR1 1.41 C hMAb TSHR1 0.92 l ng/mL 0.5 ng/mL B plus C2.08 B plus C 1.92 ¹Values shown are means of closely agreeing duplicatedeterminations

TABLE 11 Effects of NIBSC 90/672 in the cyclic AMP stimulation assayMean cyclic AMP number of Standard Sample per well (pmols)determinations Deviation Buffer only 0.60 6 0.068 0.1 units/L 1.09 60.085 0.3 units/L 1.49 5 0.11 1.0 units/L 3.52 5 0.46 3.0 units/L 8.16 61.39

TABLE 12 Inhibition of ¹²⁵I-TSH binding to TSHR coated tubes byrecombinant hMAb TSHR1 Fab expressed in 2 different E coli strains(XL1-Blue MRF′ and HB2151 cells) Culture supernatant % % Sampledilution² binding inhibition³ Assay buffer only¹ 12.1 0 UntransformedXL1- 4x 11.6 3.9 Blue MRF′ cell 8x 11.5 4.5 culture supernatant 16x 11.91.5 32x 11.9 1.5 64x 11.9 1.0 128x 12.1 −0.5 Transformed but non-induced4x 11.5 5.0 XL1-Blue MRF′ cell 8x 11.6 4.2 culture supernatant 16x 11.82.2 32x 11.1 8.4 64x 11.7 3.4 128x 11.1 8.0 Transformed and induced 4x1.5 87.4 XL1-Blue MRF′ cell 8x 2.3 81.3 culture supernatant 16x 4.7 60.932x 7.2 40.2 64x 8.9 26.4 128x 10.3 14.8 Untransformed HB2151 cell x411.2 7.3 culture supernatant x8 11.1 8.1 x16 10.9 9.7 x32 10.8 10.2 x6410.8 10.2 x128 10.6 12.3 Transformed but non- 4x 10.7 11.6 inducedHB2151 cell 8x 10.5 13.3 culture supernatant 16x 10.6 11.8 32x 10.7 11.464x 10.9 9.6 128x 10.6 11.8 Transformed and induced 4x 1.0 92.0 HB2151cell 8x 1.0 91.4 culture supernatant 16x 1.3 89.0 32x 2.2 82.0 64x 4.364.1 128x 6.7 44.8 ¹Assay buffer = 50mmol/L NaCl, 10mmol/L Tris-HCl pH7.8 ²All dilutions were in assay buffer ³ Inhibition of binding wascalculated using the formula:${\%\mspace{14mu}{inhibition}} = {100 - \left( {\frac{A}{B} \times 100} \right)}$where A = binding in the presence of test sample B = binding in thepresence of assay buffer

TABLE 13 Stimulation of cAMP production in CHO cells transfected withthe TSHR by recombinant hMAb TSHR1 Fab expressed in 2 different E colistrains (XL1-Blue MRF′ and HB2151 cells) Culture supernatant pmol/cellSample dilution² well mean × basal³ Assay buffer only¹ 0.54 0.49 1 0.44Untransformed XL1-Blue 10× 0.32 0.33 0.68 MRF′ cell culture supernatant0.35 Transformed but non-induced 10× 0.52 0.62 1.3 XL1-Blue MRF′ cellculture 0.73 supernatant 50× 0.50 0.49 0.99 0.48 Transformed and induced10× >6.4 >6.4 >13.1 XL1-Blue MRF′ cell culture >6.4 supernatant 50× 3.53.6 7.3 3.6 Untransformed HB2151 cell 10× 0.39 0.37 0.76 culturesupernatant 0.35 Transformed but non-induced 10× 0.29 0.37 0.76 HB2151cell culture 0.45 supernatant 50× 0.37 0.37 0.76 0.37 Transformed andinduced 10× >6.4 >6.4 >13.1 HB2151 cell culture >6.4 supernatant50× >6.4 >6.4 >13.1 >6.4 ¹Assay buffer = Hanks' buffered salt solution(NaCl free) containing 1 g/L glucose, 20 mmol/L HEPES, 222 mmol/Lsucrose, 15 g/L bovine serum albumin (BSA) and 0.5 mmol/L 2isobutyl-1-methyl xanthine pH 7.4 ²Dilutions in assay buffer ³Basal =cAMP produced in the presence of assay buffer only

TABLE 14 Inhibition of ¹²⁵I-TSH binding to TSHR coated tubes byrecombinant hMAb TSHR1 Fab, recombinant 4B4 Fab (a human MAb to GAD) andrecombinant hybrid Fabs (mixed HC and LC of the 2 Fabs). Expression in Ecoli HB2151 cells ASSAYS OF PERIPLASMIC FRACTIONS Periplasmic fraction(PF) dilution and (total % Fab concentration in ¹²⁵I-TSH % Test sampleundiluted PF) binding inhibition¹ Assay buffer only 11.5 0 Untransformedcells 4× (ud) 11.8 −2.7 8× 11.8 −2.7 16× 12.2 −5.9 hMAb TSHR1 HC/LC 4×(177 ng/mL) 1.6 86^(a) transformed but non- 8× 3.6 68 induced cells 16×6.4 45 hMAb TSHR1 HC/LC 4× (364 ng/mL) 0.95 92 transformed and 8× 1.4 88induced cells 16× 2.7 77 hMAb TSHRI HC/ 4× (ud) 12.0 −3.9 4B4 LCtransformed 8× 12.1 −4.8 but non-induced cells 16× 12.4 −7.8 hMAb TSHR1HC/ 4× (83 ng/mL) 11.4 0.9 4B4 LC transformed 8× 11.8 −2.1 and inducedcells 16× 11.7 −1.8 4B4 HC/hMAb TSHR1 4× (ud) 11.9 −3.5 LC transformedbut 8× 12.2 −6.1 non-induced cells 16× 12.4 −7.8 4B4 HC/hMAb TSHR1 4×(850 ng/mL) 11.8 −2.8 LC transformed and 8× 11.2 3.1 induced cells 16×11.9 −3.2 4B4 HC/LC trans- 4× (ud) 12.1 −5.4 formed but non 8× 12.0 −4.0induced cells 16× 12.1 −4.8 4B4 HC/LC trans- 4× (265 ng/mL) 11.7 −1.2formed and 8× 11.7 −1.4 induced cells 16× 12.0 −4.2 ¹Inhibition ofbinding was calculated using the formula:${\%\mspace{14mu}{inhibition}} = {100 - \left( {\frac{A}{B} \times 100} \right)}$where A = % ¹²⁵I-TSH binding in the presence of test sample B = %¹²⁵I-TSH binding in the presence of assay buffer ^(a)Detection of TSHRAb activity in the non-induced cells was due to constitutive activity ofthe promoter giving low level expression of Fab in the absence of IPTG.ud = undetectable

TABLE 15 Inhibition of ¹²⁵I-TSH binding to TSHR coated tubes byrecombinant hMAb TSHR1 Fab, recombinant 4B4 Fab (a human MAb to GAD) andrecombinant hybrid Fabs (mixed HC and LC of the 2 Fabs). Expression in Ecoli HB2151 cells ASSAYS OF CULTURE SUPERNATANTS Culture supernatantdilution and (total Fab concentration % in undiluted ¹²⁵I-TSH % Testsample supernatant) binding inhibition¹ Assay buffer only 11.1 0Untransformed cells 4× 11.8 −6.5 8× (ud) 13.1 −18 16× 11.1 −0.3 hMAbTSHR1 HC/LC 4× 10.8 2.1 transformed but 8× (ud) 12.1 −9.8 non-inducedcells 16× 11.9 −8.1 hMAb TSHR1 HC/LC 4× 1.1 91 transformed and 8× (421ng/mL) 1.2 90 induced cells 16× 1.1 90 hMAb TSHR1 HC/4B4 LC 4× 11.8 −7.0transformed but 8× (ud) 12.5 −13.0 non-induced cells 16× 12.0 −1.3 hMAbTSHR1 HC/4B4 LC 4× 10.8 2.7 transformed and 8× (262 ng/mL) 11.1 −0.4induced cells 16× 11.3 −2.6 4B4 HC/hMAb TSHR1 LC 4× 11.9 −7.4transformed but 8× (ud) 12.7 −15 non-induced cells 16× 11.8 −7.0 4B4HC/hMAb TSHR1 LC 4× 10.5 4.8 transformed and 8× (84 ng/mL) 10.8 2.4induced cells 16× 11.2 −0.9 4B4 HC/LC 4× 11.9 −7.5 transformed but 8×(ud) 12.6 −14 non-induced cells 16× 12.0 −9.0 4B4 HC/LC 4× 10.5 −4.7transformed and 8× (522 ng/mL) 11.0 0.7 induced cells 16× 11.0 0.5 ¹seefootnote to Table 14 ud = undetectable

TABLE 16 Stimulation of cyclic AMP production in CHO Cells transfectedwith the TSHR by recombinant hMAb TSHR1 Fab, recombinant 4B4 Fab (ahuman MAb to GAD) and recombinant hybrid Fabs (mixed HC and LC of the 2Fabs). Expression in E coli HB2151 cells ASSAYS OF CULTURE SUPERNATANTSDilution² of culture mean supernatant pmol pmol and (total Fab cycliccyclic concentration AMP/ AMP/ × basal in undiluted cell cell stim- Testsample¹ supernatants) well well ulation³ Assay buffer¹ only 0.35 0.31 10.25 0.33 Untransformed cells 10× 0.51 0.55 1.8 (ud) 0.67 0.48 hMAbTSHR1 HC/LC 10× 0.84 1.59 5.1^(a) transformed but (ud) 1.80 non-inducedcells 2.13 hMAb TSHR1 HC/LC 10× >6.4 >6.4 >20 transformed and (421ng/mL) >6.4 induced cells >6.4 hMAb TSHR1 HC/4B4 10× 0.55 0.59 1.9 LCtransformed but (ud) 0.63 non-induced cells 0.58 hMAb TSHR1 HC/4B4 10×0.47 0.48 1.6 LC transformed and (262 ng/mL) 0.47 induced cells 0.52 4B4HC/hMAb TSHR1 10× 0.65 0.61 2.0 LC transformed but (ud) 0.59 non-inducedcells 0.60 4B4 HC/hMAb TSHR1 10× 0.51 0.42 1.4 LC transformed and (84ng/mL) 0.37 induced cells 0.38 4B4 HC/LC 10× 0.65 0.67 2.2 transformedbut (ud) 0.73 non induced cells 0.64 4B4 HC/LC 10× 0.55 0.44 1.4transformed and (522 ng/mL) 0.41 induced cells 0.35 ¹Assay buffer:Hanks' buffered salt solution (NaCl free) containing 1 g/L glucose, 20mmol/L HEPES, 222 mmol/L sucrose, 15 g/L bovine serum albumin (BSA) and0.5 mmol/L 2 isobutyl-l-methyl xanthine pH 7.4 ²Dilutions in assaybuffer ³Basal = cyclic AMP production in the presence of assay bufferonly ^(a)Detection of cyclic AMP stimulation activity in the non-inducedcells was due to constitutive activity of the promoter giving low levelexpression of Fab in the absence of IPTG. Total recombinant Fab levelswere undetectable (detection limit = 5-10 ng/mL) whereas the stimulationof cyclic AMP assay can detect as little as 0.3 ng/mL of hMAb TSHR1 Fab.ud = undetectable

TABLE 17 Stimulation of cyclic AMP production in CHO Cells transfectedwith the TSHR by recombinant hMAb TSHR1 Fab, recombinant 4B4 Fab (ahuman MAb to GAD) and recombinant hybrid Fabs (mixed HC and LC of the 2Fabs). Expression in E coli HB2151 cells ASSAYS OF PERIPLASMIC FRACTIONSPeriplasmic fraction (PF) mean dilution² pmol pmol and total Fab cycliccyclic concentration AMP/ AMP/ × basal in undiluted cell cell stim- Testsample PF) well well ulation³ Assay buffer¹ only 0.35 0.31 1 0.25 0.33Untransformed cells 10× 0.31 0.29 0.9 (ud) 0.22 0.35 hMAb TSHR1 HC/LC10× >6.4 >6.4 >20.6^(a) transformed but (177 ng/mL) >6.4 non-inducedcells >6.4 hMAb TSHR1 HC/LC 10× >6.4 >6.4 >20.6 transformed and (364ng/mL) >6.4 induced cells — hMAb TSHR1 HC/4B4 10× 0.40 0.35 1.1 LCtransformed but (ud) 0.33 non-induced cells 0.33 hMAb TSHR1 HC/4B4 10×0.31 0.34 1.1 LC transformed and (83 ng/mL) 0.31 induced cells 0.41 4B4HC/hMAb TSHR1 10× 0.29 0.30 1.0 LC transformed but (ud) 0.31 non-inducedcells 0.29 4B4 HC/hMAb TSHR1 10× 0.23 0.24 0.8 LC transformed and (850ng/mL) 0.25 induced cells 0.24 4B4 HC/LC 10× 0.33 0.32 1.0 transformedbut (ud) 0.35 non induced cells 0.29 4B4 HC/LC 10× 0.40 0.37 1.2transformed and (265 ng/mL) 0.38 induced cells 0.32 hMAb TSHR1 IgG 2.02.0 6.4 1 ng/ml (hybridoma 2.0 produced) 2.0 ud = undetectable;^(1,2,3)see footnote to Table 16 ^(a)Detection of TSHR Ab activity inthe non-induced cells was due to constitutive activity of the promotergiving low level expression in the absence of IPTG.

TABLE 18 Inhibition of 4B4 IgG (a hybridoma produced human MAb to GAD)binding to ¹²⁵I-GAD by recombinant hMAb TSHR1 Fab, recombinant 4B4 Faband recombinant hybrid Fabs (mixed HC and LC of the 2 Fabs). Expressionin E coil HB2151 cells ASSAYS OF PERIPLASAIIC FRACTIONS Periplasmicfraction (PF) dilution and (total Fab % Test sample added withconcentration ¹²⁵I-GAD % 484 IgG and ¹²⁵I-GAD in undiluted PF) bindinginhibition¹ GAD Ab Assay buffer 28 0 4B4 F(ab')₂ 1 μg/ml 5.5 80(hybridoma 0.1 μg/ml 12 57 produced) 0.01 μg/ml 24 14 0.001 μg/ml 29−3.9 Untransformed cells 4× (ud) 27 0.9 8× 28 −1.7 16× 29 −6.3 hMAbTSHR1 HC/LC 4× (177 ng/mL) 28 −2.6 transformed but non- 8× 28 −0.5induced cells 16× 28 −0.8 hMAb TSHR1 HC/LC 4× (364 ng/mL) 27 0.7transformed and induced 8× 27 1.4 cells 16× 29 −4.2 hMAb TSHR1 HC/4B4 4×(ud) 28 −1.1 LC transformed but 8× 28 −0.2 non- induced cells 16× 27 1.1hMAb TSHR1 HC/4B4 4× (83 ng/mL) 28 −1.4 LC transformed and 8× 28 −0.7induced cells 16× 28 −2.4 4B4 HC/hMAb TSHR1 4× (ud) 28 −2.9 LCtransformed but 8× 28 −3.2 non-induced cells 16× 28 −3.1 4B4 HC/hMAbTSHR1 4× (850 ng/mL) 27 1.7 LC transformed and 8× 28 −0.2 induced cells16× 28 −1.0 4B4 HC/LC transformed 4× (ud) 29 −4.4 but non induced cells8× 28 −1.5 16× 27 1.7 4B4 HC/LC transformed 4× (265 ng/mL) 21 23.2 andinduced cells 8× 24 12.5 16× 26 5.6 ¹Inhibition of binding wascalculated using the formula:${\%\mspace{14mu}{inhibition}} = {100 - \left( {\frac{A}{B} \times 100} \right)}$where A = ¹²⁵I-GAD binding in the presence of test sample B = ¹²⁵I-GADbinding in the presence of assay buffer ud = undetectable

TABLE 19 Inhibition of 4B4 IgG (a hybridoma produced human MAb to GAD)binding to ¹²⁵I-GAD by recombinant hMAb TSHR1 Fab, recombinant 4B4 Faband recombinant hybrid Fabs (mixed HC and LC of the 2 Fabs). Expressionin E coli HB2151 ASSAYS OF CULTURE SUPERNATANTS Culture supernatantdilution and (total Fab concentration % Test sample added with inundiluted ¹²⁵I-GAD % 4B4 IgG and ¹²⁵I-GAD supernatant) bindinginhibition¹ Assay buffer 26 0 4B4 F(ab′)₂    1 μg/ml 5.2 80 (hybridoma 0.1 μg/ml 11 58 produced)  0.01 μg/ml 22 15 0.001 μg/ml 28 −5.2Untransformed 4× 28 −5.5 HB2151 cells 8× (ud) 29 −9.1 16× 28 −6.3 hMAbTSHR1 HC/LC 4× 28 −7.0 transformed but 8× (ud) 29 −9.5 non-induced cells16× 28 −5.2 hMAb TSHR1 HC/LC 4× 28 −7.0 transformed and 8× (421 ng/mL)28 −6.4 induced cells 16× 28 −5.6 hMAb TSHR1 HC/4B4 LC 4× 29 −9.0transformed but 8× (ud) 29 −7.9 non-induced cells 16× 28 −7.6 hMAb TSHR1HC/4B4 LC 4× 27 −2.2 transformed and 8× (262 ng/mL) 28 −5.5 inducedcells 16× 28 −5.1 4B4 HC/hMAb TSHR1 LC 4× 28 −4.4 transformed but 8×(ud) 28 −7.0 non-induced cells 16× 28 −5.5 4B4 HC/hMAb TSHR1 LC 4× 27−2.0 transformed and 8× (84 ng/mL) 28 −5.7 induced cells 16× 27 −2.5 4B4HC/LC 4× 28 −4.8 transformed but 8× (ud) 28 −4.8 non induced cells 16×27 −1.7 4B4 HC/LC 4× 11 59 transformed and 8× (522 ng/mL) 14 46 inducedcells 16× 17 34 ¹see footnote to Table 18 ud = undetectable

TABLE 20 Effects of mouse monoclonal antibodies to the TSHR on hMAbTSHR1 induced stimulation of cyclic AMP production in CHO cellsexpressing the TSHR mean pmol pmol cyclic cyclic AMP/ AMP/ × basal Testsample¹ cell well cell well SD stimulation² hMAb TSHR1 Fab 5 ng/mLplus:- (a) Assay buffer¹ 3.252 2.835 — 3.9 2.418 — (b) 2G2³ 4.278 3.5950.496 4.9 3.392 3.116 (c) 2B4⁴ 3.320 2.939 0.286 4.0 2.632 2.864 (d)9D33⁴ 0.506 0.443 0.047 0.61 0.394 0.428 Assay buffer alone¹ 0.696 0.7270.02 1 0.742 0.742 2G2 alone³ 0.252 0.311 0.051 0.43 0.306 0.376 2B4alone⁴ 0.298 0.331 0.033 0.46 0.318 0.376 9D33 alone⁴ 0.280 0.313 0.0250.43 0.318 0.340 ¹All dilutions were made in assay buffer (Hanks'buffered salt solution (NaCl free) containing 1 g/L glucose, 20 mmol/LHEPES, 222 mmol/L sucrose, 15 g/L bovine serum albumin (BSA) and 0.5mmol/L 2 isobutyl-1-methyl xanthine pH 7.4) ²Basal = cyclic AMPproduction in the presence of assay buffer only ³2G2 is a mousemonoclonal antibody to thyroglobulin (100 μg/mL of IgG preparation) ⁴2B4and 9D33 are mouse monoclonal antibodies to the TSHR (100 μg/mL of IgGpreparations)

TABLE 21 Effects of mouse monoclonal antibodies to the TSHR on pTSHinduced stimulation of cyclic AMP production in CHO cells expressing theTSHR pmol cyclic mean pmol × basal AMP/ cyclic AMP/ stim- Test sample¹cell well cell well SD ulation² pTSH 0.5 ng/mL plus:- (a) Assay buffer¹4.016 3.91 0.91 11.5 2.746 4.960 (b) 2B4⁴ 0.878 0.78 0.07 2.3 0.7100.742 (c) 9D33⁴ 0.436 0.43 0.01 1.3 0.436 0.410 Assay buffer alone¹0.384 0.34 0.03 1 0.318 0.318 2B4 alone⁴ 0.446 0.49 0.04 1.4 0.486 0.5529D33 alone⁴ 0.332 0.33 0.02 0.97 0.362 0.304 ^(1,2,4)See footnotes forTable 20. In a separate experiment a control mouse MAb to thyroglobulin(2G2 IgG 100 μg/mL) was shown to have no effect on pTSH (0.5 ng/mL)stimulation of cyclic AMP production (pTSH plus assay buffer = 12.7 ×basal; pTSH plus 2G2 = 11.7 × basal)

TABLE 22 Inhibition by various MAbs of ¹²⁵I-TSH, ¹²⁵I-hMAb TSHR1 or¹²⁵I- 9D33 binding to TSHR coated tubes Test IgG and Inhibition ofInhibition of concentration ¹²⁵I-TSH Inhibition of ¹²⁵I-hMAb ¹²⁵I-9D33(μg/mL)¹ binding² TSHR1 binding² binding² 9D33 IgG 0.001 0 0 4 0.01 17 39 0.1 34 21 35 1 58 44 64 10 68 56 71 2B4 IgG 0.001 15 0 4 0.01 36 0 50.1 62 0 15 1 83 11 22 10 85 18 22 hMAb 0.001 13 41 1.1 TSHR1 0.01 60 7018 IgG 0.1 89 88 72 1 94 93 90 10 95 94 93 ¹All dilutions were in a poolof healthy blood donor sera (HBD pool) ²Inhibition of binding wascalculated using the formula:${\%\mspace{14mu}{inhibition}} = {100 - \left( {\frac{A}{B} \times 100} \right)}$where A = % binding in the presence of test sample B = % binding in thepresence of HBD pool The control mouse MAb to thyroglobulin (2G20.001-100 μg/mL) had no effect on the binding of labelled TSH, hMAbTSHR1 or 9D33

TABLE 23 Effect of patient sera on ¹²⁵I-9D33 binding and ¹²⁵I-TSHbinding to TSHR coated tubes Inhibition of Inhibition of ¹²⁵I-9D33 bound¹²⁵I-9D33 ¹²⁵I-TSH Test serum¹ (% total counts added) binding (%)²binding (%)² HBD pool 11 0 0 G1 2.2 79 90 G2 4.3 74 59 G3 3.2 69 78 G45.8 45 50 G5 4.0 62 78 G6 5.1 67 51 G7 5.9 44 74 G8 2.6 75 82 G9 2.0 8190 G10 5.5 48 62 G11 3.1 62 59 G12 4.0 43 51 G13 6.0 50 59 G14 5.3 71 80G15 3.1 77 98 G16 2.4 80 93 G17 2.1 84 94 G18 1.7 73 83 G19 2.9 80 94G20 2.1 71 80 A1 10 1.9 0 A2 10 2.3 0 D1 11 0 0 D2 10 4.5 0 N1 12 −156.7 N2 7.9 25 41 N3 11 0 6.3 N4 11 −5.0 6.5 N5 9.1 14 6.3 N6 11 −2.1 7.2N7 14 −37 −1.4 N8 11 −2.1 5.9 N9 12 −9.8 6.7 ¹HBD pool = pool of healthyblood donor sera G1-G20 are sera from patients with Graves' disease D1and D2 are from patients with type 1 diabetes mellitus (positive forautoantibodies to glutamic acid decarboxylase) A1 and A2 are frompatients with Addison's disease (positive for steroid 21-hydroxylaseautoantibodies) N1-N9 are from individual healthy blood donors²Inhibition of binding was calculated using the formula:${\%\mspace{14mu}{inhibition}} = {100 - \left( {\frac{A}{B} \times 100} \right)}$where A = % binding in the presence of test serum B = % binding in thepresence of a pool of healthy blood donor sera

TABLE 24 Effect of patient sera with TSH agonist and TSH antagonistactivities on ¹²⁵I-9D33 binding to TSHR coated tubes Test sample¹²⁵I-9D33 bound Inhibition of and dilution¹ (% total counts added)¹²⁵I-9D33 binding (%)² HBD pool 11 0 Serum A 1:320 6.4 39 1:160 4.7 551:80 3.3 69 1:40 2.8 73 1:20 2.4 77 1:10 2.0 82 Serum B 1:320 9.1 131:160 8.3 21 1:80 6.4 39 1:40 4.9 53 1:20 3.8 64 1:10 2.5 76 Serum C1:320 9.7 7.6 1:160 8.9 15 1:80 8.0 24 1:40 6.3 40 1:20 5.1 51 1:10 3.765 Serum D 1:320 9.4 11 1:160 8.2 22 1:80 7.2 32 1:40 5.2 51 1:20 4.3 591:10 3.3 69 ¹HBD pool = pool of healthy blood donor sera, test sera werediluted in this pool sera A and B have TSH antagonist activity sera Cand D have TSH agonist activity ²Inhibition of binding was calculatedwith the formula used in Table 23

TABLE 25 Effect of NIBSC 90/672 on ¹²⁵I-9D33 binding and ¹²⁵I-TSHbinding to TSHR coated tubes Concentration ¹²⁵I-9D33 bound Inhibition ofInhibition of of (% total ¹²⁵I-9D33 binding² ¹²⁵I-TSH binding² 90/672¹counts added) (%) (%) 40 U/L  4.1 72 92 8 U/L 7.1 52 68 2 U/L 11 28 23 1U/L 13 10 12 0 U/L 15 0 0 ¹90/672 diluted in a pool of healthy blooddonor sera ²Inhibition of binding was calculated with the formula usedin Table 23

TABLE 26 Inhibition of ¹²⁵I-hMAb TSHR1 Fab binding to TSHR coated tubesby polyclonal hMAb TSHR1 anti idiotypic antibodies in sera from a mouseimmunised with hMAb TSHR1 Fab Dilution¹ of % inhibition² of ¹²⁵I-hMAbTSHR1 Test sample test sample Fab binding to the TSHR Assay buffer 0Sera from a mouse 1:100000 1.3 immunised with hMAb 1:50000  7.9 TSHR1Fab 1:10000  49.8 1:5000  73.0 1:1000  94.8 Non-immunised mouse 1:500  −0.8 serum ¹Test samples diluted in assay buffer. Binding in thepresence of assay buffer was 43% ²Inhibition of binding was calculatedusing the formula:${\%\mspace{14mu}{inhibition}} = {100 - \left( {\frac{A}{B} \times 100} \right)}$where A = % ¹²⁵I-hMAb TSHR1 Fab binding in the presence of test sample B= % ¹²⁵I-hMAb TSHR1 Fab binding in the presence of assay buffer

TABLE 27 Inhibition of ¹²⁵I-hMAb TSHR1 Fab binding to TSHR coated tubesby a mouse monoclonal anti idiotypic antibody 7E51 IgG % binding of %inhibition¹ of ¹²⁵I-hMAb TSHR1 ¹²⁵I-hMAb TSHR1 Test sample Fab to theTSHR Fab binding Assay buffer alone 16.3 0 7E51 IgG  1 μg/mL 14.5 11.0diluted in  10 μg/mL 6.4 60.7 assay buffer 100 μg/mL 1.7 89.4 ¹seefootnote to Table 26

1. An isolated or purified human monoclonal antibody reactive with theTSH receptor; or a fragment of a human monoclonal antibody reactive withthe TSH receptor, wherein the monoclonal antibody or fragment inhibitsTSH binding to the TSH receptor and stimulates cAMP production by cellsexpressing the TSH receptor, wherein the monoclonal antibody or fragmenthas an inhibitory activity with respect to binding of ¹²⁵I-labeled TSHto the TSH receptor of at least 15 units of International Standard NIBSC90/672 per mg.
 2. The monoclonal antibody or fragment of claim 1,wherein the monoclonal antibody or fragment has an inhibitory activityof at least 120 units of International Standard NIBSC 90/672 per mg. 3.The monoclonal antibody or fragment of claim 1, wherein the monoclonalantibody or fragment has a stimulatory activity with respect to cAMPproduction of at least 30 units of International Standard NIBSC 90/672per mg.
 4. The monoclonal antibody or fragment of claim 3, wherein themonoclonal antibody or fragment has a stimulatory activity with respectto cAMP production of at least 240 units of International Standard NIBSC90/672 per mg.
 5. The monoclonal antibody or fragment of claim 1,wherein the monoclonal antibody or fragment comprises a VH domain asshown in Seq. ID NO: 1, or one or more VH CDRs selected from SEQ ID NO:2, SEQ ID NO: 3 and SEQ ID NO:
 4. 6. The monoclonal antibody or fragmentof claim 5, wherein the monoclonal antibody or fragment furthercomprises an antibody VL domain as shown in SEQ ID NO: 6, or one or moreVL CDRs selected from SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO:
 7. Themonoclonal antibody or fragment according to claim 1, which is afragment of a human monoclonal antibody reactive with the TSH receptorand has an inhibitory activity with respect to TSH binding to the TSHreceptor of at least 30 units of International Standard NIBSC 90/672 permg.
 8. The monoclonal antibody or fragment according to claim 1, whichis a fragment of a human monoclonal antibody reactive with the TSHreceptor and has a stimulatory activity with respect to cAMP productionby cells expressing TSH of at least 50 units of International StandardNIBSC 90/672 per mg.
 9. The monoclonal antibody or fragment of claim 1,wherein the binding partner has an affinity for TSH receptor of at least10¹⁰ M⁻¹.
 10. A human recombinant antibody reactive with the TSHreceptor; or a fragment of a human recombinant antibody reactive withthe TSH receptor, wherein the recombinant antibody or fragment inhibitsTSH binding to the TSH receptor and stimulates cAMP production by cellsexpressing the TSH receptor, wherein the recombinant antibody orfragment has an inhibitory activity of at least 15 units ofInternational Standard NIBSC 90/672 per mg.
 11. The recombinant antibodyor fragment of claim 10, wherein the recombinant antibody or fragmenthas an inhibitory activity of at least 120 units of InternationalStandard NIBSC 90/672 per mg.
 12. The recombinant antibody or fragmentof claim 10, wherein the recombinant antibody or fragment has astimulatory activity with respect to cAMP production of at least 30units of International Standard NIBSC 90/672 per mg.
 13. The recombinantantibody or fragment of claim 10, wherein the monoclonal antibody orfragment has a stimulatory activity with respect to cAMP production ofat least 240 units of International Standard NIBSC 90/672 per mg. 14.The recombinant antibody or fragment of claim 10, wherein the monoclonalantibody or fragment comprises a VH domain as shown in Seq. ID NO: 1, orone or more VH CDRs selected from SEQ ID NO: 2, SEQ ID NO: 3 and SEQ IDNO:
 4. 15. The recombinant antibody or fragment of claim 10 wherein themonoclonal antibody or fragment further comprises an antibody VL domainas shown in SEQ ID NO: 6, or one or more VL CDRs selected from SEQ IDNO: 7, SEQ ID NO: 8, and SEQ ID NO:
 9. 16. The recombinant antibody orfragment of claim 10, wherein the recombinant antibody or fragment has astimulatory activity with respect to cAMP production of at least 50units of International Standard NIBSC 90/672 per mg.